US8334139B1 - Plasmids for transforming plant cells - Google Patents
Plasmids for transforming plant cells Download PDFInfo
- Publication number
- US8334139B1 US8334139B1 US06/783,336 US78333685A US8334139B1 US 8334139 B1 US8334139 B1 US 8334139B1 US 78333685 A US78333685 A US 78333685A US 8334139 B1 US8334139 B1 US 8334139B1
- Authority
- US
- United States
- Prior art keywords
- plasmid
- plant
- gene
- dna
- fragment
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000013612 plasmid Substances 0.000 title claims abstract description 509
- 230000001131 transforming effect Effects 0.000 title claims abstract description 8
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 292
- 239000003550 marker Substances 0.000 claims abstract description 29
- 244000005700 microbiome Species 0.000 claims abstract description 22
- 231100000588 tumorigenic Toxicity 0.000 claims abstract description 9
- 230000000381 tumorigenic effect Effects 0.000 claims abstract description 9
- 108010025815 Kanamycin Kinase Proteins 0.000 claims abstract description 4
- 108700039691 Genetic Promoter Regions Proteins 0.000 claims description 63
- 206010028980 Neoplasm Diseases 0.000 claims description 26
- 230000001939 inductive effect Effects 0.000 claims description 23
- 229920001184 polypeptide Polymers 0.000 claims description 19
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 19
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 19
- 241000894006 Bacteria Species 0.000 claims description 17
- 230000008488 polyadenylation Effects 0.000 claims description 12
- 241000589158 Agrobacterium Species 0.000 claims description 11
- 108091026890 Coding region Proteins 0.000 claims description 11
- 230000006870 function Effects 0.000 claims description 11
- 238000012546 transfer Methods 0.000 claims description 10
- 230000008929 regeneration Effects 0.000 claims description 8
- 238000011069 regeneration method Methods 0.000 claims description 8
- 108700001094 Plant Genes Proteins 0.000 claims description 7
- 230000006798 recombination Effects 0.000 claims description 7
- 238000005215 recombination Methods 0.000 claims description 7
- 230000003115 biocidal effect Effects 0.000 claims description 6
- 229930193140 Neomycin Natural products 0.000 claims description 2
- 229960004927 neomycin Drugs 0.000 claims description 2
- 238000010348 incorporation Methods 0.000 claims 5
- 238000001727 in vivo Methods 0.000 claims 3
- 241000589155 Agrobacterium tumefaciens Species 0.000 abstract description 49
- 238000003776 cleavage reaction Methods 0.000 abstract description 39
- 230000007017 scission Effects 0.000 abstract description 39
- 108700026220 vif Genes Proteins 0.000 abstract 1
- 239000012634 fragment Substances 0.000 description 298
- 210000004027 cell Anatomy 0.000 description 269
- 241000196324 Embryophyta Species 0.000 description 246
- 108020004414 DNA Proteins 0.000 description 156
- 238000000034 method Methods 0.000 description 101
- 108010058731 nopaline synthase Proteins 0.000 description 91
- 241000588724 Escherichia coli Species 0.000 description 53
- 239000000203 mixture Substances 0.000 description 44
- 239000013598 vector Substances 0.000 description 29
- 230000009466 transformation Effects 0.000 description 25
- 102000012410 DNA Ligases Human genes 0.000 description 24
- 108010061982 DNA Ligases Proteins 0.000 description 24
- 108010020183 3-phosphoshikimate 1-carboxyvinyltransferase Proteins 0.000 description 23
- 239000002609 medium Substances 0.000 description 23
- 102000004190 Enzymes Human genes 0.000 description 22
- 108090000790 Enzymes Proteins 0.000 description 22
- 229960000723 ampicillin Drugs 0.000 description 22
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 22
- 101150005851 NOS gene Proteins 0.000 description 21
- 238000003780 insertion Methods 0.000 description 19
- 230000037431 insertion Effects 0.000 description 19
- 108010042407 Endonucleases Proteins 0.000 description 17
- 102000004533 Endonucleases Human genes 0.000 description 17
- 230000001580 bacterial effect Effects 0.000 description 17
- 229930027917 kanamycin Natural products 0.000 description 17
- 229960000318 kanamycin Drugs 0.000 description 17
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 17
- 229930182823 kanamycin A Natural products 0.000 description 17
- 108020004999 messenger RNA Proteins 0.000 description 17
- 230000029087 digestion Effects 0.000 description 15
- 210000001938 protoplast Anatomy 0.000 description 15
- 101500006448 Mycobacterium bovis (strain ATCC BAA-935 / AF2122/97) Endonuclease PI-MboI Proteins 0.000 description 14
- 229930195732 phytohormone Natural products 0.000 description 13
- 230000008569 process Effects 0.000 description 13
- 235000018102 proteins Nutrition 0.000 description 13
- 102000004169 proteins and genes Human genes 0.000 description 13
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 12
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 108091081024 Start codon Proteins 0.000 description 12
- 239000012528 membrane Substances 0.000 description 12
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 12
- 108091028043 Nucleic acid sequence Proteins 0.000 description 11
- 108010006025 bovine growth hormone Proteins 0.000 description 11
- 239000006137 Luria-Bertani broth Substances 0.000 description 10
- 108091034117 Oligonucleotide Proteins 0.000 description 10
- 244000309466 calf Species 0.000 description 10
- 229960000268 spectinomycin Drugs 0.000 description 10
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 10
- 229930006000 Sucrose Natural products 0.000 description 9
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 9
- 239000004009 herbicide Substances 0.000 description 9
- 150000003839 salts Chemical class 0.000 description 9
- 239000005720 sucrose Substances 0.000 description 9
- 229920001817 Agar Polymers 0.000 description 8
- 244000068988 Glycine max Species 0.000 description 8
- 235000010469 Glycine max Nutrition 0.000 description 8
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 8
- 101150032520 STR gene Proteins 0.000 description 8
- 239000008272 agar Substances 0.000 description 8
- 238000010353 genetic engineering Methods 0.000 description 8
- 238000000338 in vitro Methods 0.000 description 8
- 108091008146 restriction endonucleases Proteins 0.000 description 8
- 101150070450 spc gene Proteins 0.000 description 8
- 229940088594 vitamin Drugs 0.000 description 8
- 239000011782 vitamin Substances 0.000 description 8
- 235000013343 vitamin Nutrition 0.000 description 8
- 229930003231 vitamin Natural products 0.000 description 8
- FEFNLMIFMCISIM-UHFFFAOYSA-N 1-methyl-4-nitro-5-(4-nitrophenyl)sulfanylimidazole Chemical compound CN1C=NC([N+]([O-])=O)=C1SC1=CC=C([N+]([O-])=O)C=C1 FEFNLMIFMCISIM-UHFFFAOYSA-N 0.000 description 7
- 108020004705 Codon Proteins 0.000 description 7
- LMKYZBGVKHTLTN-NKWVEPMBSA-N D-nopaline Chemical compound NC(=N)NCCC[C@@H](C(O)=O)N[C@@H](C(O)=O)CCC(O)=O LMKYZBGVKHTLTN-NKWVEPMBSA-N 0.000 description 7
- 238000010276 construction Methods 0.000 description 7
- XDDAORKBJWWYJS-UHFFFAOYSA-N glyphosate Chemical compound OC(=O)CNCP(O)(O)=O XDDAORKBJWWYJS-UHFFFAOYSA-N 0.000 description 7
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 6
- 102000053602 DNA Human genes 0.000 description 6
- 208000034454 F12-related hereditary angioedema with normal C1Inh Diseases 0.000 description 6
- 239000005562 Glyphosate Substances 0.000 description 6
- 229930195725 Mannitol Natural products 0.000 description 6
- 239000003242 anti bacterial agent Substances 0.000 description 6
- 229940088710 antibiotic agent Drugs 0.000 description 6
- 239000000499 gel Substances 0.000 description 6
- 229940097068 glyphosate Drugs 0.000 description 6
- 238000010438 heat treatment Methods 0.000 description 6
- 208000016861 hereditary angioedema type 3 Diseases 0.000 description 6
- 239000000594 mannitol Substances 0.000 description 6
- 235000010355 mannitol Nutrition 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 229960005322 streptomycin Drugs 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 102000004594 DNA Polymerase I Human genes 0.000 description 5
- 108010017826 DNA Polymerase I Proteins 0.000 description 5
- 102000003960 Ligases Human genes 0.000 description 5
- 108090000364 Ligases Proteins 0.000 description 5
- 239000011543 agarose gel Substances 0.000 description 5
- -1 aromatic amino acids Chemical class 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 238000005520 cutting process Methods 0.000 description 5
- 238000012217 deletion Methods 0.000 description 5
- 230000037430 deletion Effects 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 230000002068 genetic effect Effects 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 230000002363 herbicidal effect Effects 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 239000002773 nucleotide Substances 0.000 description 5
- 125000003729 nucleotide group Chemical group 0.000 description 5
- 230000003612 virological effect Effects 0.000 description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- 241000589156 Agrobacterium rhizogenes Species 0.000 description 4
- 241000701959 Escherichia virus Lambda Species 0.000 description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 4
- 230000000692 anti-sense effect Effects 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 229960003669 carbenicillin Drugs 0.000 description 4
- FPPNZSSZRUTDAP-UWFZAAFLSA-N carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 210000000349 chromosome Anatomy 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 4
- 230000002062 proliferating effect Effects 0.000 description 4
- 230000003362 replicative effect Effects 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- IEDVJHCEMCRBQM-UHFFFAOYSA-N trimethoprim Chemical compound COC1=C(OC)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 IEDVJHCEMCRBQM-UHFFFAOYSA-N 0.000 description 4
- 229960001082 trimethoprim Drugs 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000908115 Bolivar Species 0.000 description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 108091092195 Intron Proteins 0.000 description 3
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 241001672648 Vieira Species 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 3
- 229960005091 chloramphenicol Drugs 0.000 description 3
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 3
- 230000002779 inactivation Effects 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 108091033319 polynucleotide Proteins 0.000 description 3
- 102000040430 polynucleotide Human genes 0.000 description 3
- 239000002157 polynucleotide Substances 0.000 description 3
- 230000001172 regenerating effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- QUTYKIXIUDQOLK-PRJMDXOYSA-N 5-O-(1-carboxyvinyl)-3-phosphoshikimic acid Chemical compound O[C@H]1[C@H](OC(=C)C(O)=O)CC(C(O)=O)=C[C@H]1OP(O)(O)=O QUTYKIXIUDQOLK-PRJMDXOYSA-N 0.000 description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 108091027551 Cointegrate Proteins 0.000 description 2
- 241000195493 Cryptophyta Species 0.000 description 2
- IMXSCCDUAFEIOE-UHFFFAOYSA-N D-Octopin Natural products OC(=O)C(C)NC(C(O)=O)CCCN=C(N)N IMXSCCDUAFEIOE-UHFFFAOYSA-N 0.000 description 2
- IMXSCCDUAFEIOE-RITPCOANSA-N D-octopine Chemical compound [O-]C(=O)[C@@H](C)[NH2+][C@H](C([O-])=O)CCCNC(N)=[NH2+] IMXSCCDUAFEIOE-RITPCOANSA-N 0.000 description 2
- 108010066133 D-octopine dehydrogenase Proteins 0.000 description 2
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 2
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 2
- 244000000626 Daucus carota Species 0.000 description 2
- 235000002767 Daucus carota Nutrition 0.000 description 2
- 108010005054 Deoxyribonuclease BamHI Proteins 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 241001524679 Escherichia virus M13 Species 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 108010070675 Glutathione transferase Proteins 0.000 description 2
- 102000005720 Glutathione transferase Human genes 0.000 description 2
- 108010051696 Growth Hormone Proteins 0.000 description 2
- 241000208818 Helianthus Species 0.000 description 2
- 235000003222 Helianthus annuus Nutrition 0.000 description 2
- 101150062179 II gene Proteins 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- 108010050904 Interferons Proteins 0.000 description 2
- 102000014150 Interferons Human genes 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 108020005120 Plant DNA Proteins 0.000 description 2
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 2
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 2
- 108020004682 Single-Stranded DNA Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 102100038803 Somatotropin Human genes 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 238000000376 autoradiography Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000031018 biological processes and functions Effects 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 238000012364 cultivation method Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000001627 detrimental effect Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 239000000122 growth hormone Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 229940125396 insulin Drugs 0.000 description 2
- 229940079322 interferon Drugs 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical compound OS(O)(=O)=O.O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 description 2
- 229960002064 kanamycin sulfate Drugs 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 239000002777 nucleoside Substances 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- UZKQTCBAMSWPJD-UQCOIBPSSA-N trans-Zeatin Natural products OCC(/C)=C\CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-UQCOIBPSSA-N 0.000 description 2
- UZKQTCBAMSWPJD-FARCUNLSSA-N trans-zeatin Chemical compound OCC(/C)=C/CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-FARCUNLSSA-N 0.000 description 2
- 230000005026 transcription initiation Effects 0.000 description 2
- 238000000844 transformation Methods 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
- 239000001226 triphosphate Substances 0.000 description 2
- 235000011178 triphosphate Nutrition 0.000 description 2
- 229940023877 zeatin Drugs 0.000 description 2
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 description 1
- HCWLJSDMOMMDRF-SZWOQXJISA-N 3-[(3s,6r)-2-oxo-6-[(1s,2r,3r)-1,2,3,4-tetrahydroxybutyl]morpholin-3-yl]propanamide Chemical compound NC(=O)CC[C@@H]1NC[C@H]([C@@H](O)[C@H](O)[C@H](O)CO)OC1=O HCWLJSDMOMMDRF-SZWOQXJISA-N 0.000 description 1
- 241001135511 Agrobacterium rubi Species 0.000 description 1
- ILQOASSPNGAIBC-UHFFFAOYSA-N Agropine Natural products OC(O)C(O)CC(O)C1CN2C(CCC2=O)C(=O)O1 ILQOASSPNGAIBC-UHFFFAOYSA-N 0.000 description 1
- 240000003291 Armoracia rusticana Species 0.000 description 1
- 241001167018 Aroa Species 0.000 description 1
- 108700003860 Bacterial Genes Proteins 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 101000583080 Bunodosoma granuliferum Delta-actitoxin-Bgr2a Proteins 0.000 description 1
- 108090000565 Capsid Proteins Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 102100023321 Ceruloplasmin Human genes 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- YAHZABJORDUQGO-NQXXGFSBSA-N D-ribulose 1,5-bisphosphate Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)C(=O)COP(O)(O)=O YAHZABJORDUQGO-NQXXGFSBSA-N 0.000 description 1
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 description 1
- 108010047524 Deoxyribonuclease HindIII Proteins 0.000 description 1
- 101100491986 Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139) aromA gene Proteins 0.000 description 1
- 241001131785 Escherichia coli HB101 Species 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 101710138460 Leaf protein Proteins 0.000 description 1
- 241000209510 Liliopsida Species 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 241000218922 Magnoliophyta Species 0.000 description 1
- 241000283956 Manis Species 0.000 description 1
- 240000004658 Medicago sativa Species 0.000 description 1
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 description 1
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 1
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 1
- 208000005289 Neoplastic Cell Transformation Diseases 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 240000007377 Petunia x hybrida Species 0.000 description 1
- 108010064851 Plant Proteins Proteins 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 108010056088 Somatostatin Proteins 0.000 description 1
- 102000005157 Somatostatin Human genes 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical group O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 241001464837 Viridiplantae Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 239000002647 aminoglycoside antibiotic agent Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 101150037081 aroA gene Proteins 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 239000007844 bleaching agent Substances 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 244000038559 crop plants Species 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 230000003544 deproteinization Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 235000021186 dishes Nutrition 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Natural products O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000012606 in vitro cell culture Methods 0.000 description 1
- 239000003617 indole-3-acetic acid Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 238000007169 ligase reaction Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 230000006680 metabolic alteration Effects 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 239000003471 mutagenic agent Substances 0.000 description 1
- LCNBIHVSOPXFMR-UHFFFAOYSA-N n'-(3-aminopropyl)butane-1,4-diamine;hydron;trichloride Chemical compound Cl.Cl.Cl.NCCCCNCCCN LCNBIHVSOPXFMR-UHFFFAOYSA-N 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 231100001221 nontumorigenic Toxicity 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000031787 nutrient reservoir activity Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- 210000000745 plant chromosome Anatomy 0.000 description 1
- 235000021118 plant-derived protein Nutrition 0.000 description 1
- 238000013492 plasmid preparation Methods 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 108010055896 polyornithine Proteins 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002250 progressing effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 101150079601 recA gene Proteins 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 229910052701 rubidium Inorganic materials 0.000 description 1
- IGLNJRXAVVLDKE-UHFFFAOYSA-N rubidium atom Chemical compound [Rb] IGLNJRXAVVLDKE-UHFFFAOYSA-N 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- YZHUMGUJCQRKBT-UHFFFAOYSA-M sodium chlorate Chemical compound [Na+].[O-]Cl(=O)=O YZHUMGUJCQRKBT-UHFFFAOYSA-M 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 229960000553 somatostatin Drugs 0.000 description 1
- NHXLMOGPVYXJNR-ATOGVRKGSA-N somatostatin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N1)[C@@H](C)O)NC(=O)CNC(=O)[C@H](C)N)C(O)=O)=O)[C@H](O)C)C1=CC=CC=C1 NHXLMOGPVYXJNR-ATOGVRKGSA-N 0.000 description 1
- 235000019710 soybean protein Nutrition 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 239000010455 vermiculite Substances 0.000 description 1
- 235000019354 vermiculite Nutrition 0.000 description 1
- 229910052902 vermiculite Inorganic materials 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
Definitions
- This invention is in fields of genetic engineering, plant biology, and bacteriology.
- plant refers to a multicellular differentiated organism that is capable of photosynthesis, such as angiosperms and multicellular algae. This does not include microorganisms, such as bacteria, yeast, and fungi.
- plant cell includes any cell derived from a plant; this includes undifferentiated tissue such as callus or crown gall tumor, as well as plant seeds, propagules, pollen, or plant embryos.
- T-DNA The tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens has been proposed for use as a natural vector for introducing foreign genetic information into plant cells (Hernalsteen et al 1980; Rorsch and Schilperoort, 1978). Certain types of A. tumefaciens are capable of infecting a wide variety of plant cells, causing crown gall disease. The process of infection is not fully understood. At least part of the Ti plasmid enters the plant cell. Various metabolic alterations occur, and part of the Ti plasmid is inserted into the genome of the plant (presumably into the chromosomes). The part of the Ti plasmid that enters the plant genome is designated as “transferred DNA” (T-DNA). T-DNA is stably maintained in the plant DNA (Chilton et al, 1977; Yadev et al, 1980; Willmitzer et al, 1980; Otten et al, 1981).
- a different species of Agrobacterium carries a “root-inducing” (Ri) plasmid which is similar to the Ti plasmid. Infection of a plant cell by A. rhizogenes causes hairy root disease. Like the Ti plasmid, a segment of DNA called “T-DNA” (also referred to by some researchers as “R-DNA”) is transferred into the plant genome of an infected cell.
- Ri root-inducing
- plant tumor inducing plasmid includes any plasmid (1) which is contained in a microorganism, other than a virus, which is capable of causing genetic transformation of one or more types of plants or plant cells, and (2) which contains a segment of DNA which is inserted into a plant genome. This includes Ri plasmids.
- T-DNA refers to a segment of DNA in or from Plant tumor inducing plasmid (1) which has been inserted into the genome of one or more types of plant cells, or (2) which is contained in a segment of DNA that is located between two sequences of bases which are capable of serving as T-DNA borders.
- T-DNA border and border are determined and applied empirically; these terms shall refer to a sequence of bases which appears at or near the end of a segment of DNA which is transferred from a plant tumor inducing plasmid into a plant genome.
- the T-DNA which is inserted into and expressed in plant cells, contains genes which are involved in the production of high levels of phytohormones in the transformed plant cells (Leemans et al 1982).
- the high levels of phytohormones interfere with the normal metabolic and regenerative process of the cells, and prevent the formation of phenotypically normal plants from the cells (Braun and Wood, 1976; Yang et al, 1980). Exceptions to this are rare cases where the T-DNA has undergone extensive spontaneous deletions in planta to eliminate those genes involved in phytohormone production. Under these conditions, normal plants are reported to be obtainable at low frequency (Otten et al, 1981).
- the T-DNA genes involved in phytohormone production could not be deleted prior to this invention, since they were very important in the identification and/or selection of transformed plant cells (Marton et al, 1979):
- the intermediate vectors contain relatively small subfragments of the Ti plasmid which can be manipulated using standard recombinant DNA techniques.
- the subfragments can be modified by the deletion of specific sequences or by the insertion of foreign genes at specific sites.
- the intermediate vectors containing the modified T DNA subfragment are then introduced into A. tumefaciens by transformation or conjugation.
- Double recombination between the modified T-DNA fragment on the intermediate vector and its wild-type counterpart on the Ti plasmid results in the replacement of the wild-type copy with the modified fragment.
- Cells which contain the recombined Ti plasmids can be selected using appropriate antibiotics.
- a major disadvantage of the above methods is that the frequency of double recombination is quite low, about 10 ⁇ 4 to 10 ⁇ 9 (Leemans et al, 1981) and it requires extensive effort to identify and isolate the rare double-crossover recombinants. As a result, the number and types of experiments which can be performed using existing methods for genetically engineering the Ti plasmid is severely limited.
- lipid vesicles also called liposomes
- the liposomes and their DNA contents may be taken up by plant cells; see, e.g., Lurquin, 1981. If the inserted DNA can be incorporated into the plant genome, replicated, and inherited, the plant cells will be transformed.
- Another method has been developed involving the fusion of bacteria, which contain desired plasmids, with plant cells. Such methods involve converting the bacteria into spheroplasts and converting the plant cells into protoplasts. Both of these methods remove the cell wall barrier from the bacterial and plant cells, using enzymic digestion. The two cell types can then be used together by exposure to chemical agents, such as polyethylene glycol. See Hasezawa et al, 1981.
- a gene which is inserted into a plant cell will not be stably maintained by the plant cell unless it is incorporated into the genome of the plant cell, i.e., unless the gene is inserted into a chromosome or plasmid that replicates in the plant cell.
- This invention relates to several plasmids which are useful for creating transformed plant cells which are capable of subsequent regeneration into differentiated, morphologically-normal plants.
- This invention also relates to microorganisms containing such plasmids, and to methods for creating such plasmids and microorganisms.
- This invention involves a first plasmid, such as pMON120, which has certain desired characteristics described below.
- a gene which is capable of being expressed in plant cells may be inserted into this plasmid to obtain a derivative plasmid, such as pMON128.
- plasmid pMON128 contains a chimeric gene which expresses neomycin phosphotransferase II (NPT II), an enzyme which inactivates certain antibiotics.
- NPT II neomycin phosphotransferase II
- the chimeric gene is capable of expression in plant cells.
- the derivative plasmid is inserted into a suitable microorganism, such as Agrobacterium tumefaciens cells which contain plant tumor inducing plasmids.
- Agrobacterium tumefaciens cells which contain plant tumor inducing plasmids.
- some of the inserted plasmids recombine with, plant tumor inducing plasmids to form a co-integrate plasmid; this is due to a region of homology between the two plasmids. Only a single crossover event is required to create the desired co-integrate plasmid.
- the resulting co-integrate plant tumor inducing contain the chimeric gene and/or any other inserted gene within the T-DNA region of the co-integrate plasmid.
- the inserted gene(s) are surrounded by at least two T-DNA borders, at least one of which was inserted into the plant tumor inducing plasmid by the crossover event.
- A. tumefaciens cells with co-integrate plasmids are co-cultured with plant cells, such as protoplasts, protoplast-derived cells, plant cuttings, or intact plants, under conditions which allow the co-integrate plant tumor inducing plasmids, or portions thereof, to enter the plant cells.
- plant cells such as protoplasts, protoplast-derived cells, plant cuttings, or intact plants
- plant cells such as protoplasts, protoplast-derived cells, plant cuttings, or intact plants
- a gene which codes for an enzyme, which inactivates a herbicide may be inserted into a plant.
- a gene which codes for a desired mammalian polypeptide such as growth hormone, insulin, interferon, or somatostatin may be inserted into plants. The plants may be grown and harvested, and the polypeptide could be extracted from the plant tissue.
- FIG. 1 is a flow chart indicating the steps of this invention, using pMON128 and the NOS-NPT II gene as an example.
- FIG. 2 represents the creation of pMON41, a plasmid used to construct pMON120.
- FIG. 3 represents the creation of M-4, an M13-derived DNA used to construct pMON109.
- FIG. 4 represents the creation of pMON54, a plasmid used to construct pMON109.
- FIG. 5 represents the creation of pMON109, a plasmid, used to construct pMON120.
- FIG. 6 represents the creation of pMON113, a plasmid used to construct pMON120.
- FIG. 7 represents the creation of plasmid pMON120, an intermediate vector with three restriction endonuclease cleavage sites which are suitable for the insertion of a desired gene.
- FIG. 8 represents the creation of pMON128, an intermediate vector which was obtained by inserting a chimeric NOS-NPT II kanamycin-resistance gene into pMON120.
- FIG. 9 represents the cointegration of pMON128 with a wild-type Ti plasmid by means of a single crossover event, thereby creating a co-integrate plasmid with multiple borders.
- FIG. 10 indicates the co-integration of pMON128 with a disarmed Ti-plasmid, thereby creating a non-tumorigenic cointegrate plasmid.
- FIG. 11 is a graph comparing growth of transformed cells and non-transformed cells on kanamycin-containing medium.
- FIG. 12 represents the structure of a typical eukaryotic gene.
- FIG. 13 is a flow chart representing steps of this invention, correlated with an example chimeric NOS-NPTII-NOS gene
- FIG. 14 represents fragment HindIII-23, obtained by digesting a Ti plasmid with HindIII.
- FIG. 15 represents a DNA fragment which contains a NOS promoter region, a NOS 5′ non-translated region, and the first few codons of the NOS structural sequence.
- FIG. 16 represents the cleavage of a DNA sequence at a precise location, to obtain a DNA fragment which contains a NOS promoter region and complete 5′ non-translated region.
- FIG. 17 represents the creation of plasmids pMON1001 and pMON40, which contain an NPTII structural sequence.
- FIG. 18 represents the insertion of a NOS promoter region into plasmid pMON40, to obtain pMON58.
- FIG. 19 represents the creation of an M13 derivative designated as M-2, which contains a NOS 3′ non-translated region and poly-A signal.
- FIG. 20 represents the assembly of the NOS-NPTII-NOS chimeric gene, and the insertion of the chimeric gene into plasmid pMON38 to obtain plasmids pMON75 and pMON76.
- FIG. 21 represents the creation of plasmid pMON66, which contains an NPTI gene.
- FIG. 22 represents the creation of plasmid pMON73, containing a chimeric NOS-NPTII sequence.
- FIG. 23 represents the creation of plasmid pMON78, containing a chimeric NOS-NPTI sequence.
- FIG. 24 represents the creation of plasmids pMON106 and pMON107, which contain chimeric NOS-NPTI-NOS genes.
- FIG. 25 represents the insertion of a chimeric NOS-NPTI-NOS gene into pMON120 to obtain plasmids pMON130 and pMON131.
- FIG. 26 represents the structure of a DNA fragment containing a soybean protein (sbss) promoter.
- FIG. 27 represents the creation of plasmid pMON121, containing the sbss promoter.
- FIG. 28 represents the insertion of a chimeric sbss-NPTII-NOS gene into pMON120 to create plasmids pMON141 and pMON142.
- FIG. 29 represents the creation of plasmid pMON108, containing a bovine growth hormone structural sequence and a NOS 3′ region.
- FIG. 30 represents the creation of plasmid N25-BGH, which contains the BGH-NOS sequence surrounded by selected cleavage sites.
- FIG. 31 represents the insertion of a chimeric sbss-BGH-NOS gene into pMON120 to obtain plasmids pMON147 and pMON148.
- FIG. 32 represents the creation of plasmid pMON149, which contains a chimeric NOS-BGH-NOS gene.
- FIG. 33 represents the creation of plasmid pMON8, which contains a structural sequence for EPSP synthase.
- FIG. 34 represents the creation of plasmid pMON25, which contains an EPSP synthase structural sequence with several cleavage site near the start codon.
- FIG. 35 represents the creation of plasmid pMON146, which contains a chimeric sequence comprising EPSP synthase and a NOS 3′ region.
- FIG. 36 represents the insertion of a chimeric NOS-EPSP-NOS gene into pMON120 to obtain plasmid pMON153.
- FIG. 37 represents the creation of plasmid pMON154, which contains a chimeric sbss-EPSP-NOS gene.
- a variety of chimeric genes were inserted into plant cells using the steps that are summarized on the flow chart of FIG. 1 . As shown on FIG. 1 , three preliminary plasmids were prepared. Those plasmids were designated as:
- pMON41 which contained a right border from a nopaline-type Ti plasmid, and the 5′ portion of a nopaline synthase (NOS) gene.
- NOS nopaline synthase
- pMON109 which contained the 3′ portion of a NOS gene, and a selectable marker gene (spc/str) which allowed for the selection of A. tumefaciens cells having co-integrate Ti plasmids with chimeric genes.
- spc/str selectable marker gene
- pMON113 which contained a region of DNA with a sequence that is identical to the sequence within the T-DNA portion of an octopine-type Ti plasmid. This region was designated as the “left inside homology” (LIR) region. The construction of pMON113 is described below and shown in FIG. 6 .
- each plasmid was digested by appropriate endonucleases to obtain a desired fragment.
- Three fragments (one from each of the three plasmids) were assembled in a triple ligation to obtain the intermediate vector, plasmid pMON120, as shown in FIG. 7 .
- Plasmid pMON120 plays a key role in the embodiment of this invention that is described in detail below. This plasmid has the following characteristics:
- pMON120 has at least three unique restriction endonuclease cleavage sites (EcoRI, ClaI, and HindIII) which allow for the convenient insertion of any desired gene.
- pMON120 will replicate within normal E. coli cells. However, it will not replicate within normal Agrobacterium cells unless it co-integrates with another plasmid, such as a Ti plasmid, which will replicate in Agrobacterium cells.
- pMON120 carries a marker gene which codes for an enzyme which confers resistance to two antibiotics, spectinomycin (spc) and streptomycin (str). This gene, referred to as the spc/str gene, is expressed in E. coli and in A. tumefaciens , but not in plant cells. pMON120 does not carry genes which code for resistance to ampicillin or tetracycline.
- pMON120 carries a sequence which is homologous to a sequence within the T-DNA portion of an octopine-type Ti plasmid of A. tumefaciens . This sequence is referred to as the “left inside homology” (LIH) region. This region of homology promotes a crossover event whereby pMON120, or a derivative of pMON120 such as pMON128, forms a co-integrate with the Ti plasmid if the two plasmids exist inside the same A. tumefaciens cell.
- the “co-integrate” plasmid is formed by a single crossover event. It contains all DNA sequences that previously existed in either the Ti plasmid or the pMON120-derived plasmid.
- pMON120 carries a nopaline-type T-DNA “right border,” i.e., a sequence which is capable of acting as one end (designated by convention as the right border) of a T-DNA sequence which is transferred from a Ti plasmid and inserted into the chromosome of a plant cell during transformation of the cell by A. tumefaciens.
- pMON120 carries a gene (including a promoter) which codes for the expression of an enzyme, nopaline synthase (NOS). Once introduced into a plant cell, the NOS enzyme catalyzes the production of nopaline, a type of opine. In most types of plants, opines are non-detrimental compounds which accumulate at low levels; the presence of nopaline can be readily detected in plant tissue (Otten and Schilperoort, 1978). Opine genes may serve as useful marker genes to confirm transformation, since opines do not normally exist in untransformed plant cells.
- the NOS gene in pMON120 may be rendered non-functional by a variety of techniques known to those skilled in the art. For example, a BamHI cleavage site exists within the coding portion of the NOS gene; a stop codon or other appropriate oligonucleotide sequence could be inserted into this site to prevent the translation of NOS.
- the modified T-DNA region only part of the co-integrate Ti plasmid (the modified T-DNA region) will be inserted into the plant genome. Therefore, only a part of the pMON120-derived plasmid will be inserted into the plant genome. This portion begins at the T-DNA border, and stretches in one direction only to the region of homology. In pMON120, the NOS scorable marker, the spc/str selectable marker, and the three insertion sites are within the portion of pMON120 that would be transferred into the plant genome. However, the pBR322-derived region next to the LIH, and the PvuI cleavage site, probably would not be transferred into the plant genome. Importantly, this arrangement of pMON120 and its derivatives prevents the transfer of more than one region of homology into the plant genome, as discussed below.
- pMON120 is about 8 kilobases long. This is sufficiently small to allow it to accomplish all of the objectives of this invention. However, if desired, it may be made somewhat smaller by the deletion of one or more nucleotide sequences which are not essential, using methods which are known to those skilled in the art. Such a reduction in size might improve the efficiency or other characteristics of the plasmid when used for this invention or for other purposes, as may be determined by those skilled in the art.
- the NOS marker used for scoring transformed plant cells might be deleted, or replaced or supplemented by a different scorable or selectable marker.
- One such marker gene might comprise an antibiotic-resistance gene such as the NOS-NPT II-NOS chimeric gene described below.
- the spc/str marker gene used for selecting A. tumefaciens cells with co-integrate plasmids might be deleted, or replaced or supplemented by a different scorable or selectable marker that is expressed in Agrobacteria .
- T-DNA borders such as a nopaline-type “left” border, or an octopine-type left or right border
- more than one border such as two or more nopaline right borders, or one nopaline right border and one octopine right border
- both left and right borders of any type
- any type of desired plasmid such as a nopaline or agropine Ti plasmid or an Ri plasmid; such regions will allow the intermediate vector to form a co-integrate with any desired plasmid.
- Plasmid pMON120 was constructed from fragments derived from 3 other plasmids. These three plasmids were designated as pMON41, pMON109, and pMON113. The construction of each of these three plasmids is summarized below; additional information is provided in the examples.
- Plasmid pMON41 contributed a nopaline-type T-DNA right border and the 5′ portion of a nopaline synthase (NOS) gene to pMON120. It was created by the following method.
- a nopaline-type Ti plasmid designated as the pTiT37 plasmid, may be digested with the HindIII endonuclease to produce a variety of fragments, including a 3.4 kb fragment which is designated as the HindIII-23 fragment. This fragment contains the entire NOS gene and the T-DNA right border.
- the Applicants inserted a HindIII-23 fragment into a plasmid, pBR327 (Soberon et al, 1980), which had been digested with HindIII.
- the resulting plasmid designated as pMON38, was digested with both HindIII and BamHI.
- This 2.3 kb fragment was inserted into a pBR327 plasmid which had been digested with HindIII and BamHI.
- the resulting plasmid was designated as pMON41, as shown in FIG. 2 .
- a variety of strains of A. tumefaciens are publicly available from the American Type Culture Collection (Rockville, Mb): accession numbers are listed in any ATCC catalog. Each strain contains a Ti plasmid which is likely to be suitable for use in this invention, as may be determined through routine experimentation by those skilled in the art.
- Plasmid pMON109 contributed a spc/str selectable marker gene and the 3′ portion of a NOS gene to pMON120. It was created by the following method.
- Plasmid pMON38 (described above and shown on FIG. 2 ) was digested with RsaI, which creates blunt ends as shown: 5′-GTAC CATG
- a 1.1 kb fragment was isolated, and digested with BamHI to obtain fragments of 720 bp and 400 bp, each of which had one blunt Rsa end and a cohesive BamHI end. These fragments were added to double stranded DNA from a phage M13 mp 8 (Messing and Vieira, 1982) which had been digested with SmaI (which creates blunt ends) and BamHI. The mixture was ligated, transformed into cells and plated for recombinant phage. Recombinant phage DNA's which contained the inserted 720 bp fragment were identified by the size of the BamHI-SmaI insert. One of those phage DNA's was designated as M-4, as shown in FIG.
- the 720 bp insert contained the 3′ non-translated region (including the poly-adenylation signal indicated in the figures by a heavy dot) of the NOS gene, as well as the 3′ portion of the structural sequence of the NOS gene.
- the 720 bp insert is surrounded in M-4 by EcoRI and PstI cleavage sites, which were present in the M13 mp 8 DNA.
- a bacterial transposon designated as Tn7
- Tn7 A bacterial transposon, designated as Tn7
- the Tn7 transposon also contains a gene which causes the host cell to be resistant to the antibiotic trimethoprim.
- the exact location and orientation of the spc/str gene and the trimethoprim-resistance gene in Tn7, are not known.
- the Tn7 transposon may be obtained from a variety of cell strains which are publicly available.
- a strain of A. tumefaciens was isolated in which the Tn7 transposon had been inserted into the Hind 111-23 region of a pTiT37 plasmid.
- the modified pTiT37 plasmid was designated as pGV3106 (Hernalsteens et al, 1980).
- Plasmid pGV3106 was digested with HindIII, and the fragments were shotgun-cloned into pBR327 plasmids which had been digested with HindIII. These plasmids were inserted into E. coli cells, and cells which were ampicillin-resistant (due to a pBR327 gene) and trimethoprim-resistant (due to a Tn7 gene) were selected. The plasmid obtained from one colony was designated as pMON31. This plasmid contained a 6 kb HindIII insert. The insert contained the spc/str-resistance gene and trimethoprim-resistance gene from Tn7, and the 3′ portion of a NOS gene (which came from the pTiT37 plasmid).
- Plasmid pMON31 was reduced in size twice. The first reduction was performed by digesting the plasmid with EcoRI, diluting the mixture to remove an 850 bp fragment, and religating the large fragment. The resulting plasmid, designated as pMON53, was obtained from transformed cells selected by their resistance to ampicillin and streptomycin. Resistance to trimethoprim was not determined.
- Plasmid pMON53 was further reduced in size by digesting the plasmid with ClaI, diluting the mixture to remove a 2 kb fragment, and religating the large fragment.
- the resulting 5.2 kb plasmid was designated as pMON54, as shown in FIG. 4 .
- This plasmid contains the spc/str gene.
- Plasmid pMON54 was digested with EcoRI and PstI, and a 4.8 kb fragment containing the spc/str gene was isolated. M-4 DNA was digested with EcoRI and PstI, and a 740 bp fragment containing the NOS 3′ non-translated region was isolated. These fragments were ligated together to form pMON64.
- the orientation of the spc/str gene was reversed by digesting pMON64 with ClaI and religating the mixture. Plasmids having the desired orientation were identified by cleavage using EcoRI and BamHI. These plasmids were designated as pMON109, as shown in FIG. 5 .
- Plasmid pMON113 contributed a region of homology to pMON120 which allows pMON120 to form a co-integrate plasmid when present in A. tumefaciens along with a Ti plasmid.
- the region of homology was taken from an octopine-type Ti plasmid. In the Ti plasmid, it is located near the left T-DNA border, within the T-DNA portion of the Ti plasmid. This region of homology is designated as the “left inside homology” (L1H) region.
- a region of homology may be derived from any type of plasmid capable of transforming plant cells, such as any Ti plasmid or any Ri plasmid.
- An intermediate vector can be designed which can form a co-integrate plasmid with whatever type of plasmid the region of homology was derived from.
- the region of homology it might not be necessary for the region of homology to be located within the T-DNA.
- a region of homology may be derived from a segment of a Ti plasmid Which contains a T-DNA border and a sequences of bases outside of the T-DNA region.
- the intermediate vector contains two appropriate T-DNA borders, it might be possible for the region of homology to be located entirely outside of the T-DNA region.
- the Bam-8 fragment which is about 7.5 kb, contains the left border and the LIH region of the Ti plasmid (Willmitzer et al, 1982; DeGreve et al, 1981).
- the Bam-8 fragment was inserted into the plasmid pBR327, which had been digested with BamHI.
- the resulting plasmid was designated as pMON90, as shown in FIG. 6 .
- Plasmid pMON90 was digested with BglII, and a 2.6 kb fragment which contains the LIH region but not the left border was purified.
- the 2.6 kb fragment was treated with Klenow polymerase to convert the cohesive ends into blunt ends, and the fragment was digested with HindIII to obtain a 1.6 kb fragment (the desired fragment) and a 1 kb fragment.
- Both fragments were mixed with a pBR322 plasmid which had been digested with PvuII and HindIII. The mixture was ligated, and inserted into E. coli cells.
- the cells were selected for ampicillin resistance, and scored for the presence of a SmaI site which exists on the 1.6 kb fragment but not the 1 kb fragment.
- a colony having the desired plasmid was identified, and the plasmid froth this colony was designated as pMON113, as indicated by FIG. 6 .
- Plasmid pMON41 was digested with PvuI and BamI, and a 1.5 kb fragment containing a nopaline-type right border and the 5′ portion of a NOS gene was isolated.
- Plasmid pMON109 was digested with BamHI and EcoRI, and a 3.4 kb fragment containing a spc/str gene and the 3′ part of a NOS gene was isolated.
- Plasmid pMON113 was digested with PvuI and EcoRI, and a 3.1 kb fragment containing the LIH region was isolated.
- a culture of E. coli containing pMON120 has been deposited with the American Type Culture Center. This culture has been assigned accession number 39263.
- pMON120 a variety of different methods could be used to create pMON120, or any similar intermediate vector. For example, instead of the triple ligation, it would have been possible to assemble two of the desired fragments in a plasmid, and insert the third fragment into the plasmid.
- pMON120 has three unique cleavage sites (EcoRI, ClaI, and HindIII) which are suitable for the insertion of any desired gene. These cleavage sites are located in the portion of pMON120 that will be inserted into a plant genome, so the inserted gene also will be inserted into the plant genome.
- chimeric genes which are capable of expressing bacterial and mammalian polypeptides in plant cells have been created by the Applicants. These chimeric genes are described in detail in a separate, simultaneously-filed application entitled “Chimeric Genes Suitable for Expression in Plant Cells,” Ser. No. 458,414. The contents of that application are hereby incorporated by reference. Those chimeric genes are suitable for use in this invention. They may be inserted into pMON120 to create a derivative plasmid, which may be utilized as described below.
- the chimeric gene comprises a promoter region which is capable of causing RNA polymerase in a plant cell to create messenger RNA corresponding to the DNA.
- One such promoter region comprises a nopaline synthase (NOS) promoter region, which normally exists in certain types of Ti plasmids in bacteria, A. tumefaciens .
- NOS nopaline synthase
- the NOS gene normally is inactive while contained in A. tumefaciens cells, and it becomes active after the Ti plasmid enters a plant cell.
- Other suitable promoter regions may be derived from genes which exist naturally in plant cells.
- the chimeric gene also contains a sequence of bases which codes for a 5′ non-translated region of mRNA which is capable of enabling or increasing the expression in a plant cell of a structural sequence of the mRNA.
- a suitable 5′ non-translated region may be taken from the NOS gene mentioned above, or from a gene which exists naturally in plant cells.
- the chimeric gene also contains a desired structural sequence, i.e., a sequence which is transcribed into mRNA which is capable of being translated into a desired polypeptide.
- the structural sequence is heterologous with respect to the promoter region, and it may code for any desired polypeptide, such as a bacterial or mammalian protein.
- the structural sequence includes a start codon and a stop codon.
- the structural sequence may contain introns which are removed from the mRNA prior to translation.
- the chimeric gene may also contain a DNA sequence which codes for a 3′ non-translated region (including a poly-adenylation signal) of mRNA.
- This region may be derived from a gene which is naturally expressed in plant cells, to help ensure proper expression of the structural sequence.
- genes include the NOS gene mentioned above, as well as genes which exist naturally in plant cells.
- a chimeric gene of this invention will be expressed in the plant cell to create a desired polypeptide, such as a mammalian hormone, or a bacterial enzyme which confers antibiotic or herbicide resistance upon the plant.
- a chimeric gene which comprises the following DNA sequences:
- NPT II neomycin phosphotransferase II
- This chimeric NOS-NPT II-NOS gene was isolated on a DNA fragment having EcoRI ends. This fragment was inserted into the EcoRI cleavage site of pMON120, and the resulting plasmids (having chimeric gene inserts with opposite orientations) were designated as pMON128 and pMON129, as shown in FIG. 8 . Plasmid pMON129 has two copies of the chimeric gene; this may be a useful feature in certain types of work. Either plasmid may be utilized to transform plant cells, in the following manner. A culture of E. coli containing pMON128 has been deposited with the American Type Culture Collection. This culture has been assigned accession number 39264.
- NOS nopaline synthase
- the NOS is normally carried in certain types of Ti plasmids, such as pTiT37. Sciaky et al, 1978.
- the NOS promoter is normally inactive while in an A. tumefaciens cell.
- the entire NOS gene, including the promoter and the protein coding sequence, is within the T-DNA portion of a Ti plasmid that is inserted into the chromosomes of plant cells when a plant becomes infected and forms a crown gall tumor.
- the NOS promoter region directs RNA polymerase within a plant cell to transcribe the NOS protein coding sequence into mRNA, which is subsequently translated into the NOS enzyme.
- a promoter region shown in FIG. 12 as association region 2 , intervening region 4 , transcription initiation sequence 6 , and intervening region 8 ), and the boundary between the promoter region and the 5′ non-translated region, are not fully understood.
- the Applicants decided to utilize the entire promoter region and 5′ non-translated region from the NOS gene, which is known to be expressed in plant cells. However, it is entirely possible that one or more of these sequences might be modified in various ways, such as alteration in length or replacement by other sequences.
- a nopaline-type tumor-inducing plasmid designated as pTiT37, was isolated from a strain of A. tumefaciens using standard procedures (Currier and Nester, 1976). It was digested with the endonuclease HindIII which produced numerous fragments. These fragments were separated by size on a gel, and one of the fragments was isolated and removed from the gel. This fragment was designated as the HindIII-23 fragment, because it was approximately the 23rd largest fragment from the Ti plasmid; it is approximately 3400 base pairs (bp) in size, also referred to as 3.4 kilobases (kb).
- the HindIII-23 fragment contained the entire NOS gene, including the promoter region, a 5′ non-translated region, a structural sequence with a start codon and a stop codon, and a 3′ non-translated region.
- the HindIII-23 fragment is shown in FIG. 14 .
- the HindIII-23 fragment could be digested by another endonuclease, Sau3a, to yield a fragment, about 350 bp in size, which contains the entire NOS promoter region, the 5′ non-translated region, and the first few codons of the NOS structural sequence.
- This fragment was sequenced, and the base sequence is represented in FIG. 15 .
- the start codon (ATG) of the NOS structural sequence begins at base pair 301 within the 350 bp fragment.
- the Applicants decided to cleave the fragment between base pairs 300 and 301; this would provide them with a fragment about 300 base pairs long containing a NOS promoter region and the entire 5′ non-translated region but with no translated bases.
- an M13 clone designated as S1A, and utilized the procedure described below.
- Dr. Michael Bevan of Washington University converted the 350 bp Sau3a fragment into a single strand of DNA. This was done by utilizing a virus vector, designated as the M13 mp 2 phage, which goes through both double-stranded (ds) and single-stranded (ss) stages in its life cycle (Messing et al, 1981).
- the ds 350 bp fragment was inserted into the double-stranded replicative form DNA of the M13 mp 2, which had been cleaved with BamHI. The two fragments were ligated, and used to infect E. coli cells.
- the ds DNA containing the 350 bp inserted fragment subsequently replicated, and one strand (the viral strand) was encapsulated by the M13 viral capsid proteins.
- the orientation of the 350 bp fragment was such that the anti-sense strand (containing the same sequence as the mRNA) of the NOS gene was carried in the viral strand.
- Viral particles released from infected cells were isolated, and provided to the Applicants.
- Single stranded S1A DNA containing the anti-sense 350 bp fragment with the NOS promoter region, was isolated from the viral particles and sequenced.
- a 14-mer oligonucleotide primer was synthesized, using published procedures (Beaucage and Carruthers, 1981, as modified by Adams et al, 1982). This 14-mer was designed to be complementary to bases 287 through 300 of the 350 bp fragment, as shown on FIG. 15 .
- the 5′ end of the synthetic primer was radioactively labelled with 32 P; this is represented in the figures by an asterisk
- the partially double-stranded DNA was then digested by a third endonuclease, HaeIII, which can cleave both single-stranded and double-stranded DNA.
- HaeIII cleavage sites were known to exist in several locations outside the 350 bp insert, but none existed inside the 350 bp insert. This created a fragment having one blunt end, and one 3′ overhang which started at base #301 of the Sau3a insert.
- the HaeIII fragment mixture was treated with T4 DNA polymerase and unlabelled dNTP's. This caused the single stranded portion of the DNA, which extended from base #301 of the Sau3a insert to the closest HaeIII cleavage site, to be removed from the fragment. In this manner, the ATG start codon was removed from base pair #300, leaving a blunt end double-stranded fragment which was approximately 550 bp long.
- the mixture was then digested by a fourth endonuclease EcoRI, which cleaved the 550 bp fragment at a single site outside the NOS promoter region.
- the fragments were then separated by size on a gel, and the radioactively-labelled fragment was isolated.
- This fragment contained the entire NOS promoter region and 5′ non-translated region. It had one blunt end with a sequence of 5′ . . . CTGCA . . . GACGT and one cohesive end (at the EcoRI site) with a sequence of The shorter strand was about 308 bp long.
- steps 42 , 44 , and 46 are represented in FIG. 2 as steps 42 , 44 , and 46 .
- This fragment was inserted into pMON40 (which is described below) to obtain pMON58, as shown on FIG. 13 .
- a bacterial transposon designated as Tn5
- Tn5 A bacterial transposon, designated as Tn5
- the NPTII enzyme inactivates certain aminoglycoside antibiotics, such as kanamycin, neomycin, and G418; see Jimenez and Davies, 1980.
- This gene is contained within a 1.8 kb fragment, which can be obtained by digesting phage lambda bbkan-1 DNA (D. Berg et al, 1975) with two endonucleases, HindIII and BamHI.
- This fragment was inserted into a common laboratory plasmid, pBR327, which had been digested by HindIII and BamHI.
- the resulting plasmid was designated as pMON1001, which was about 4.7 kb.
- the Applicants eliminated about 500 bp from the pMON1001 plasmid, in the following manner. First, they digested pMON1001 at a unique SmaI restriction site which was outside of the NPTII gene. Next, they inserted a 10-mer synthetic oligonucleotide linker, 5′CCGGATCCGG, GGCCTAGGCC into the SmaI cleavage site. This eliminated the SmaI cleavage site and replaced it with a BamHI cleavage site. A second BamHI cleavage site already existed, about 500 bp from the new BamHI site.
- the resulting plasmids were inserted into E. coli , which were then selected for resistance to ampicillin and kanamycin.
- a clonal colony of E. coli was selected; these cells contained a plasmid which was designated as pMON40, as shown in FIG. 17 .
- steps 48 and 50 are represented in FIG. 13 as steps 48 and 50 .
- the polymerase and dNTP's were removed, and the cleaved plasmid was then digested with EcoRI.
- the smaller fragment which contained the NPTII promoter region was removed, leaving a large fragment with one EcoRI end and one blunt end. This large fragment was mixed with the 308 bp fragment which contained the NOS promoter, described previously and shown on FIG. 5 .
- the fragments were ligated, and inserted into E. coli.
- E. coli clones were selected for ampicillin resistance.
- Replacement of the NPTII promoter region (a bacterial promoter) with the NOS promoter region (which is believed to be active only in plant cells) caused the NPTII structural sequence to become inactive in E. coli . Plasmids from 36 kanamycin-sensitive clones were obtained; the plasmid from one clone, designated as pMON58, was utilized in subsequent work.
- steps 52 and 54 are represented in FIG. 13 as steps 52 and 54 .
- Plasmid pMON58 may be digested to obtain a 1.3 kb EcoRI-BamHI fragment which contains the NOS promoter region, the NOS 5′ non-translated region, and the NPTII structural sequence. This step is represented in FIG. 13 as step 56 .
- the Applicants decided to add a NOS 3′ non-translated region to the chimeric gene, in addition to the NPTII 3′ non-translated region already present. Whether a different type of 3′ non-translated region (such as a 3′ region from an octopine-type or agropine-type Ti plasmid, or a 3′ region from a gene that normally exists in a plant cell) would be suitable or preferable for use in any particular type of chimeric gene, for use in any specific type of plant cell, may be determined by those skilled in the art through routine experimentation using the method of this invention. Alternately, it is possible, using the methods described herein, to delete the NPTII or other existing 3′ non-translated region and replace it with a desired 3′ non-translated region that is known to be expressed in plant cells.
- the Applicants utilized a 3.4 kb HindIII-23 fragment from a Ti plasmid, shown on FIG. 14 .
- This 3.4 kb fragment was isolated and digested with BamHI to obtain a 1.1 kb BamHI-HindIII fragment containing a 3′ portion of the NOS structural sequence (including the stop codon), and the 3′ non-translated region of the NOS gene (including the poly-adenylation signal).
- This 1.1 kb fragment was inserted into a pBR327 plasmid which had been digested with HindIII and BamHI.
- the resulting plasmid was designated as pMON42, as shown on FIG. 19 .
- Plasmid pMON42 was digested with BamHI and RsaI, and a 720 bp fragment containing the desired NOS 3′ non-translated region was purified on a gel.
- the 720 bp fragment was digested with another endonuclease, MboI, and treated with the large fragment of E. coli DNA polymerase I. This resulted in a 260 bp fragment with MboI blunt ends, containing a large part of the NOS 3′ non-translated region including the poly-A signal.
- step 58 The foregoing procedure is represented in FIG. 13 by step 58 .
- alternate means could have been utilized; for example, it might have been possible to digest the HindIII-23 fragment directly with MboI to obtain the desired 260 bp fragment with the NOS 3′ non-translated region.
- the Applicants decided to convert the MboI ends of the 260 bp fragment into a BamHI end (at the 5′ end of the fragment) and an EcoRI end (at the 3′ end of the fragment). In order to perform this step, the Applicants used the following method.
- the 260 bp MboI fragment was inserted into M13 mp 8 DNA at a SmaI cleavage site.
- the SmaI site is surrounded by a variety of other cleavage sites present in the M13 mp 8 DNA, as shown in FIG. 19 .
- the MboI fragment could be inserted into the blunt SmaI ends in either orientation.
- the orientation of the MboI fragments in different clones were tested, using HinfI cleavage sites located assymetrically within the MboI fragment.
- a clone was selected in which the 3′ end of the NOS 3′ non-translated region was located near the EcoRI cleavage site in the M13 mp 8 DNA. This clone was designated as the M-2 clone, as shown in FIG. 19 .
- Replicative form (double stranded) DNA from the M-2 clone was digested by EcoRI and BamHI and a 280 bp fragment was isolated. Separately, plasmid pMON58 was digested by EcoRI and BamHI, and a 1300 bp fragment was isolated. The two fragments were ligated, as shown in FIG. 20 , to complete the assembly of a NOS-NPTII-NOS chimeric gene having EcoRI ends.
- the two EcoRI-BamHI fragments could be joined together with DNA ligase and cleaved with EcoRI.
- a vector molecule having EcoRI ends that were treated with calf alkaline phosphatase (CAP) may be added to the mixture.
- the fragments in the mixture may be ligated in a variety of orientations.
- the plasmid mixture is used to transform E. coli , and cells having plasmids with the desired orientation are selected or screened, as described below.
- Plasmid pMON38 A plasmid, designated as pMON38, was created by insertion of the HindIII-23 fragment (from Ti plasmid pTiT37) into the HindIII cleavage site of the plasmid pBR327. Plasmid pMON38 contains a unique EcoRI site, and an ampicillin-resistance gene which is expressed in E. coli . Plasmid pMON38 was cleaved with EcoRI and treated with alkaline phosphatase to prevent it from re-ligating to itself. U.S. Pat. No. 4,264,731 (Shine, 1981).
- the resulting fragment was mixed with the 1300 bp NOS-NPTII fragment from pMON58, and the 280 bp NOS fragment from M-2, which had been ligated and EcoRI-cleaved as described in the previous paragraph.
- the fragments were ligated, and inserted into E. coli .
- the E. coli cells which had acquired intact plasmids with ampicillin-resistance genes were selected on plates containing ampicillin.
- Several clones were selected, and the orientation of the inserted chimeric genes was evaluated by means of cleavage experiments. Two clones having plasmids carrying NOS-NPTII-NOS inserts with opposite orientations were selected and designated as pMON75 and pMON76, as shown in FIG. 20 .
- the chimeric gene may be isolated by digesting either pMON75 or pMON76 with EcoRI and purifying a 1580 bp fragment.
- step 60 The foregoing procedure is represented on FIG. 13 by step 60 .
- NOS-NPTII-NOS chimeric gene This completes the discussion of the NOS-NPTII-NOS chimeric gene. Additional information on the creation of this gene is provided in the Examples. A copy of this chimeric gene is contained in plasmid pMON128; it may be removed from pMON128 by digestion with EcoRI. A culture of E. coli containing pMON128 has been deposited with the American Type Culture Collection; this culture has been assigned accession number 39264.
- NPTII structural sequence was expressed in the plant cells, causing them and their descendants to acquire resistance to concentrations of kanamycin which are normally toxic to plant cells.
- a chimeric gene comprising (1) a NOS promoter region and 5′ non-translated region, (2) a structural sequence which codes for NPTI, and (3) a NOS 3′ non-translated region.
- NPTI and NPTII are different and distinct enzymes with major differences in their amino acid sequences and substrate specificities. See, e.g., E. Beck et al, 1982. The relative stabilities and activities of these two enzymes in various types of plant cells are not yet fully understood, and NPTI may be preferable to NPTII for use in certain types of experiments and plant transformations.
- a 1200 bp fragment containing an entire NPTI gene was obtained by digesting pACYC177 (Chang and Cohen, 1978) with the endonuclease, AvaII.
- the Avail termini were converted to blunt ends with Klenow polymerase, and converted to BamHI termini using a synthetic linker.
- This fragment was inserted into a unique BamHI site in a pBR327-derived plasmid, as shown in FIG. 21 .
- the resulting plasmid was designated as pMON66.
- Plasmid pMON57 (a deletion derivative of pBR327, as shown in FIG. 21 ) was digested with AvaII. The 225 bp fragment of pMON57 was replaced by the analogous 225 bp AvaII fragment taken from plasmid pUC8 (Vieira and Messing, 1982), to obtain a derivative of pMON57 with no PstI cleavage sites. This plasmid was designated as pMON67.
- Plasmid pMON58 (described previously and shown in FIG. 18 ) was digested with EcoRI and BamHI to obtain a 1300 bp fragment carrying the NOS promoter and the NPTII structural sequence.
- This fragment was inserted into pMON67 which had been digested with EcoRI and BamHI.
- the resulting plasmid was designated as pMON73, as shown in FIG. 22 .
- pMON73 was digested with PstI and BamHI, and a 2.4 kb fragment was isolated containing a NOS promoter region and 5′ non-translated region.
- Plasmid pMON66 (shown on FIG. 21 ) was digested with XhoI and BamHI to yield a 950 bp fragment containing the structural sequence of NPTI. This fragment lacked about 30 nucleotides at the 5′ end of the structural sequence.
- the pMON73 fragment, the pMON66 fragment, and the synthetic linker were ligated together to obtain plasmid pMON78, as shown in FIG. 13 .
- This plasmid contains the NOS promoter region and 5′ non-translated region adjoined to the NPTI structural sequence. The ATG start codon was in the same position that the ATG start codon of the NOS structural sequence had occupied
- Plasmid pMON78 was digested with EcoRI and BamHI to yield a 1300 bp fragment carrying the chimeric NOS-NPTI regions.
- Double-stranded DNA from the M-2 clone (described previously and shown on FIG. 20 ) was digested with EcoRI and BamHI, to yield a 280 bp fragment carrying a NOS 3′ non-translated region with a poly-adenylation signal.
- the two fragments described above were ligated together to create the NOS-NPTI-NOS chimeric gene, which was inserted into plasmid pMON38 (described above) which had been digested with EcoRI.
- the two resulting plasmids, having chimeric gene inserts with opposite orientations, were designated as pMON106 and pMON107, as shown in FIG. 24 .
- Either of plasmids pMON106 or pMON107 may be digested with EcoRI to yield a 1.6 kb fragment containing the chimeric NOS-NPTI-NOS gene. This fragment was inserted into plasmid pMON120 which had been digested with EcoRI and treated with alkaline phosphatase. The resulting plasmids, having inserts with opposite orientations, were designated as pMON130 and pMON131, as shown on FIG. 25 .
- the NOS-NPTI-NOS chimeric gene was inserted into plant cells, which acquired resistance to kanamycin. This demonstrates expression of the chimeric gene in plant cells.
- a chimeric gene comprising (1) a promoter region and 5′ non-translated region taken from a gene which naturally exists in soybean; this gene codes for the small subunit of ribulose-1,5-bis-phosphate carboxylase (sbss for soybean small subunit); (2) a structural sequence which codes for NPTII, and (3) a NOS 3′ non-translated region.
- the sbss gene codes for a protein in soybean leaves which is involved in photosynthetic carbon fixation.
- the sbss protein is the most abundant protein in soybean leaves (accounting for about 10% of the total leaf protein), so it is likely that the sbss promoter region causes prolific transcription.
- ss RuBPCase protein there are believed to be approximately six genes encoding the ss RuBPCase protein in the soybean genome.
- SRS1 which is highly transcribed in soybean leaves, has been cloned and characterized.
- the promoter region, 5′ nontranslated region, and a portion of the structural sequence are contained on a 2.1 kb EcoRI fragment that was subcloned into the EcoRI site of plasmid pBR325 (Bolivar, 1978).
- the resultant plasmid, pSRS2.1 was a gift to Monsanto Company from Dr. R. B. Meagher, University of Georgia, Athens, Ga.
- the 2.1 kb EcoRI fragment from pSRS2.1 is shown on FIG. 26 .
- Plasmid pSRS2.1 was prepared from dam— E. coli cells, and cleaved with MboI to obtain an 800 bp fragment. This fragment was inserted into plasmid pKC7 (Rao and Rogers, 1979) which had been cleaved with BglII. The resulting plasmid was designated as pMON121, as shown on FIG. 27 .
- Plasmid pMON121 was digested with EcoRI and Bcl1, and a 1200 bp fragment containing the sbss promoter region was isolated. Separately, plasmid pMON75 (described previously and shown on FIG. 20 ) was digested with EcoRI and BglII, and a 1250 bp fragment was isolated, containing a NPTII structural sequence and a NOS 3′ non-translated region. The two fragments were ligated at the compatible Bcl1/BglII overhangs, to create a 2450 bp fragment containing sbss-NPTII-NOS chimeric gene.
- This fragment was inserted into pMON120 which had been cleaved with EcoRI, to create two plasmids having chimeric gene inserts with opposite orientations, as shown in FIG. 28 .
- the plasmids were designated as pMON141 and pMON142.
- the sbss-NPTII-NOS chimeric genes were inserted into several types of plant cells, causing the plant cells to acquire resistance to kanamycin.
- the chimeric sbss-NPTII-NOS gene also had another significant feature. Sequencing experiments indicated that the 800 bp MboI fragment contained the ATG start codon of the sbss structural sequence. Rather than remove this start codon, the Applicants decided to insert a stop codon behind it in the same reading frame.
- the sbss promoter is contained in plasmid pMON154, described below.
- a culture of E. coli containing this plasmid has been deposited with the American Type Culture Center. This culture has been assigned accession number 39265.
- a structural sequence which codes for the polypeptide, bovine growth hormone, was inserted into a pBR322-derived plasmid.
- the resulting plasmid was designated as plasmid CP-1.
- This plasmid was digested with EcoRI and HindIII to yield a 570 bp fragment containing the structural sequence.
- Double stranded M-2 RF DNA (described previously and shown in FIG. 19 ) was cleaved with EcoRI and HindIII to yield a 290 bp fragment which contained the NOS 3′ non-translated region with a poly-adenylation signal.
- the two fragments were ligated together and digested with EcoRI to create an 860 base pair fragment with EcoRI ends, which contained a BGH-coding structural sequence joined to the NOS 3′ non-translated region.
- This fragment was introduced into plasmid pMON38, which had been digested with EcoRI and treated with alkaline phosphatase, to create a new plasmid, designated as pMON108, as shown in FIG. 29 .
- a unique BglII restriction site was introduced at the 5′ end of the BGH structural sequence by digesting pMON 108 with EcoRI to obtain the 860 bp fragment, and using Klenow polymerase to create blunt ends on the resulting EcoRI fragment.
- This fragment was ligated into plasmid N25 (a derivative of pBR327 containing a synthetic linker carrying BglII and XbaI cleavage sites inserted at the BamHI site), which had been cleaved with XbaI and treated with Klenow polymerase to obtain blunt ends (N25 contains a unique BglII site located 12 bases from the XbaI site).
- the resulting plasmid which contained the 860 bp BGH-NOS fragment in the orientation shown in FIG. 30 , was designated as plasmid N25-BGH.
- This plasmid contains a unique BglII cleavage site located about 25 bases from the 5′ end of the BGH structural sequence.
- Plasmid N25-BGH prepared from dam— E. coli cells was digested with BglII and ClaI to yield an 860 bp fragment which contained the BGH structural sequence joined to the NOS 3′ non-translated region.
- plasmid pMON121 (described previously and shown in FIG. 27 ) was prepared from dam— E. coli cells and was digested with ClaI and BclI to create an 1100 bp fragment which contained the sbss promoter region. The fragments were ligated at their compatible BclI/BglII overhangs, and digested with ClaI to yield a ClaI fragment of about 2 kb containing the chimeric sbss-BGH-NOS gene.
- This fragment was inserted into pMON 120 (described previously and shown in FIG. 8 ) which had been digested with ClaI.
- the resulting plasmids, containing the inserted chimeric gene in opposite orientations were designated pMON 147 and pMON 148, as shown in FIG. 31 .
- An alternate chimeric BGH gene was created which contained (1) a NOS promoter region and 5′ non-translated region, (2) a structural sequence which codes for BGH, and (3) a NOS 3′ non-translated region, by the following method, shown in FIG. 32 .
- Plasmid pMON76 (described above and shown in FIG. 20 ) was digested with EcoRI and BglII to obtain a 308 bp fragment containing a NOS promoter region and 5′ non-translated region.
- Plasmid N25-BGH prepared from dam— E. coli cells (described above and shown in FIG. 30 ) was digested with BglII and ClaI to obtain a 900 bp fragment containing a BGH structural sequence and a NOS 3′ non-translated region. These two fragments were ligated together to obtain a chimeric NOS-BGH-NOS gene in a fragment with EcoRI and ClaI ends. This fragment was ligated with an 8 kb fragment obtained by digesting pMON120 with EcoRI and ClaI. The resulting plasmid, designated as pMON149, is shown in FIG. 32 .
- a chimeric gene comprising (1) a NOS promoter region and 5′ non-translated region, (2) a structural sequence which codes for the E. coli enzyme, 5-enolpyruvylshikimate-3-phosphoric acid synthase (EPSP synthase) and (3) a NOS 3′ non-translated region.
- E. coli enzyme 5-enolpyruvylshikimate-3-phosphoric acid synthase (EPSP synthase)
- ESP synthase 5-enolpyruvylshikimate-3-phosphoric acid synthase
- EPSP synthase is believed to be the target enzyme for the herbicide, glyphosate, which is marketed by Monsanto Company under the registered trademark, “Roundup.” Glyphosate is known to inhibit EPSP synthase activity (Amrhein et al, 1980), and amplification of the EPSP synthase gene in bacteria is known to increase their resistance to glyphosate. Therefore, increasing the level of EPSP synthase activity in plants may confer resistance to glyphosate in transformed plants. Since glyphosate is toxic to most plants, this provides for a useful method of weed control. Seeds of a desired crop plant which has been transformed to increase EPSP synthase activity may be planted in a field. Glyphosate may be applied to the field at concentrations which will kill all non-transformed plants, leaving the non-transformed plants unharmed.
- An EPSP synthase gene may be isolated by a variety of means, including the following.
- a lambda phage library may be created which carries a variety of DNA fragments produced by HindIII cleavage of E. coli DNA. See, e.g., Maniatis et al, 1982.
- EPSP synthase gene is one of the genes which are involved in the production of aromatic amino acids. These genes are designated as the “aro” genes; EPSP synthase is designated as aroA. Cells which do not contain functional aro genes are designated as aro-cells. Aro ⁇ cells must normally be grown on media supplemented by aromatic amino acids. See Pittard and Wallis, 1966.
- Different lambda phages which carry various HindIII fragments may be used to infect mutant E. coli cells which do not have EPSP synthase genes.
- the infected aro ⁇ cells may be cultured on media which does not contain the aromatic amino acids, and transformed aro+ clones which are capable of growing on such media may be selected. Such clones are likely to contain the EPSP synthase gene.
- Phage particles may be isolated from such clones, and DNA may be isolated from these phages.
- the phage DNA may be cleaved with one or more restriction endonucleases, and by a gradual process of analysis, a fragment which contains the EPSP synthase gene may be isolated.
- the Applicants isolated an 11 kb HindIII fragment which contained the entire E. coli EPSP synthase gene. This fragment was digested with BglII to produce a 3.5 kb HindIII-BglII fragment which contained the entire EPSP synthase gene. This 3.5 kb fragment was inserted into plasmid pKC7 (Rao and Rogers, 1979) to produce plasmid pMON4, which is shown in FIG. 33 .
- Plasmid pMON4 was digested with ClaI to yield a 2.5 kb fragment which contained the EPSP synthase structural sequence. This fragment was inserted into pBR327 that had been digested with ClaI, to create pMON8, as shown in FIG. 33 .
- pMON8 was digested with BamHI and NdeI to obtain a 4.9 kb fragment. This fragment lacked about 200 nucleotides encoding the amino terminus of the EPSP synthase structural sequence.
- the missing nucleotides were replaced by ligating a HinfI/NdeI fragment, obtained from pMON8 as shown in FIG. 34 , together with a synthetic oligonucleotide sequence containing (1) the EPSP synthase start codon and the first three nucleotides, (2) a unique BglII site, and (3) the appropriate BamHI and HinfI ends.
- the resulting plasmid, pMON25 contains an intact EPSP synthase structural sequence with unique BamHI and BglII sites positioned near the start codon.
- Double stranded M-2 DNA (described previously and shown in FIG. 19 ) was digested with HindIII and EcoRI to yield a 290 bp fragment which contains the NOS 3′ non-translated region and poly-adenylation signal. This fragment was introduced into a pMON25 plasmid that had been digested with EcoRI and HindIII to create a plasmid, designated as pMON146 (shown in FIG. 35 ) which contains the EPSP structural sequence joined to the NOS 3′ non-translated region.
- pMON146 was cleaved with ClaI and BglII to yield a 2.3 kb fragment carrying the EPSP structural sequence joined to the NOS 3′ non-translated region.
- pMON76 (described previously and shown in FIG. 20 ) was digested with BglII and EcoRI to create a 310 bp fragment containing the NOS promoter region and 5′ non-translated region.
- the above fragments were mixed with pMON 120 (described previously and shown in FIG. 8 ) that had been digested with ClaI and EcoRI, and the mixture was ligated.
- the resulting plasmid, designated pMON153 is shown in FIG. 36 . This plasmid contains the chimeric NOS-EPSP-NOS gene.
- Plasmid pMON146 (described previously and shown in FIG. 35 ) was digested with ClaI and BglII, and a 2.3 kb fragment was purified. This fragment contained the EPSP synthase structural sequence coupled to a NOS 3′ non-translated region with a poly-adenylation signal.
- Plasmid pMON121 (described above and shown in FIG. 27 ) was digested with ClaI and BclI and a 1.1 kb fragment was purified. This fragment contains an sbss promoter region and 5′ non-translated region.
- the two fragments were mixed and ligated with T4 DNA ligase and subsequently digested with ClaI.
- This chimeric gene with ClaI termini was inserted into plasmid pMON120 which had been digested with ClaI and treated with calf alkaline phosphatase (CAP).
- CAP calf alkaline phosphatase
- the mixture was ligated with T4 DNA ligase.
- the resulting mixture of fragments and plasmids was used to transform E. coli cells, which were selected for resistance to spectinomycin.
- a colony of resistant cells was isolated, and the plasmid in this colony was designated as pMON154, as shown in FIG. 37 .
- a culture of E. coli containing pMON154 has been deposited with the American Type Culture Center. This culture has been assigned accession number 39265.
- Plasmid pMON128 (or any other plasmid derived by inserting a desired gene into pMON120) is inserted into a microorganism which contains an octopine-type Ti plasmid (or other suitable plasmid).
- Suitable microorganisms include A. tumefaciens and A. rhizogenes which carry Ti or Ri plasmids.
- Other microorganisms which might also be useful for use in this invention include other species of Agrobacterium , as well as bacteria in the genus Rhizobia . The suitability of these cells, or of any other cells known at present or hereafter discovered or created, for use in this invention may be determined through routine experimentation by those skilled in the art.
- the plasmid may be inserted into the microorganism by any desired method, such as transformation (i.e., contacting plasmids with cells that have been treated to increase their uptake of DNA) or conjugation with cells that contain the pMON128 or other plasmids.
- the inserted plasmid (such as pMON128) has a region which is homologous to a sequence within the Ti plasmid.
- This “LIH” region of homology allows a single crossover event whereby pMON128 and an octopine-type Ti plasmid combine with each other to form a co-integrate plasmid. See, e.g., Stryer, supra, at p. 752-754. Normally, this will occur within the A. tumefaciens cell after pMON128 has been inserted into the cell.
- the co-integrate plasmid may be created in a different type of cell or in vitro, and then inserted into an A. tumefaciens or other type of cell which can transfer the co-integrate plasmid into plant cells.
- the inserted plasmid such as pMON128, combines with the Ti plasmid in the manner represented by FIG. 9 or 10 , depending upon what type of Ti plasmid is involved.
- item 2 represents the T-DNA portion of an octopine-type Ti plasmid.
- Item 4 represents the inserted plasmid, such as pMON128.
- Co-integrate plasmid 6 has one left border 8 , and two right borders 10 and 12 .
- the two right borders are designated herein as the “proximal” right border 10 (the right border closest to left border 8 ), and the “distal” right border 12 (the right border that is more distant from left border 8 .
- Proximate right border 10 was carried by plasmid 4 ; the distal right border was contained on Ti plasmid 2 before co-integration.
- a culture of A. tumefaciens GV3111 containing a co-integrate plasmid formed by pMON1-28 and wild-type Ti plasmid pTiB653 has been deposited with the American Type Culture Center. This culture has been assigned accession number 39266.
- T-DNA region 14 is bounded by left border 8 and proximate right border 10 .
- Region 14 contains the chimeric gene and any other genes contained in plasmid 4 , such as the spc/str selectable marker and the NOS scorable marker. However, region 14 does not contain any of the T-DNA genes which would cause crown gall disease or otherwise disrupt the metabolism or regenerative capacity of the plant cell.
- T-DNA region 16 contains left border 8 and both right borders 10 and 12 . This segment of T-DNA contains the chimeric gene and any other genes contained in plasmid 4 . However, T-DNA region 16 also contains the T-DNA genes which are believed to cause crown gall disease.
- T-DNA segments Region 14 or Region 16
- Either of the foregoing T-DNA segments, Region 14 or Region 16 might be transferred to the plant DNA. This is presumed to occur at a 50-50 probability for any given T-DNA transfer. This is likely to lead to a mixture of transformed cells, some of which are tumorous and some or which are non-tumorous. It is possible to isolate and cultivate non-tumorous cells from the mixture, as described in the examples.
- a Ti or Ri plasmid may be disarmed by one or more of the following types of mutations:
- pAL4421 One such disarmed octopine plasmid, which has a left border but not a right border, is designated as pAL4421; this plasmid is contained in A. tumefaciens strain LBA4421 (Ooms et al, 1982; Garfinkel et al, 1981).
- FIG. 10 represents an octopine-type Ti plasmid with a T-DNA region 22 which undergoes mutation to delete the tms and tmr genes and the right border. This results in a disarmed Ti plasmid with partial T-DNA region 24 .
- plasmid 26 such as pMON1228
- a crossover event occurs which creates a co-integrate Ti plasmid with disarmed T-DNA region 28 .
- the LIH region of homology is repeated in this Ti plasmid, but the disarmed Ti plasmid does not contain any oncogenic genes.
- the tms and tmr genes and the octopine synthase (OCS) gene would be contained in the co-integrate disarmed Ti plasmid; however, they would have been located outside of the T-DNA borders.
- the disarmed co-integrate Ti plasmid is used to infect plant cells, and T-DNA region 28 enters the plant genome, as shown by transformed DNA 30. Plant cells transformed by disarmed T-DNA 28 have normal phytohormone metabolism, and normal capability to be regenerated into differentiated plants.
- Plasmid pMON120 and its derivatives contain a marker gene (spc/str), which is expressed in A. tumefaciens . However, these plasmids do not replicate in A. tumefaciens . Therefore, the spc/str marker gene will not be replicated or stably inherited by A.
- A. tumefaciens cells which contain this co-integrate plasmid can readily be identified and selected by growth of the cells on medium containing either spc or str, or both.
- the Ti plasmid 28 shown in FIG. 10 , contains two LIH regions. It is possible that co-integrate Ti plasmids will undergo a subsequent crossover event, wherein the two LIH regions will recombine. This event is undesirable, since it can lead to a deletion of the DNA between the LIH regions, including the chimeric gene. However, this is not likely to lead to serious difficulties, for two reasons. First, this event is likely to occur at a relatively low probability, such as about 10 ⁇ 2 . Second, plasmid pMON120 and its derivatives, have been designed so that the selectable marker gene (spc/str) is located in the region of DNA that would be deleted by the crossover event. Therefore, the selective conditions that are used to identify and culture Agrobacteria cells containing co-integrate plasmids will also serve to kill the descendants of cells that undergo a subsequent crossover event which eliminates the chimeric gene from the Ti plasmid.
- spc/str selectable marker gene
- the plant cells to be transformed are contacted with enzymes which remove the cell walls. This converts the plant cells into protoplasts, which are viable cells surrounded by membranes. The enzymes are removed, and the protoplasts begin to regenerate cell wall material.
- the A. tumefaciens cells (which contain the co-integrate Ti plasmids with chimeric genes) are mixed with the plant protoplasts. The cells are co-cultivated for a period of time which allows the A. tumefaciens to infect the plant cells. After an appropriate co-cultivation period, the A. tumefaciens cells are killed, and the plant cells are propagated.
- Plant cells which have been transformed i.e., cells which have received DNA from the co-integrate Ti plasmids
- Plant cells which have received DNA from the co-integrate Ti plasmids may be selected by a variety of methods, depending upon the type of gene(s) that were inserted into the plant genome.
- certain genes may cause various antibiotics to be inactivated; such genes include the chimeric NOS-NPT II-NOS gene carried by pMON128.
- Such genes may serve as selectable markers; a group of, cells may be cultured on medium containing the antibiotic which is inactivated by the chimeric gene product, and only those cells containing the selectable marker gene will survive.
- genes may serve as scorable markers in plant cells.
- pMON120 and its derivative plasmids, such as pMON128, carry a nopaline synthase (NOS) gene which is expressed in plant cells.
- NOS nopaline synthase
- This gene codes for an enzyme which catalyzes the formation of nopaline.
- Nopaline is a non-detrimental compound which usually is accumulated at low quantities in most types of plants; it can be easily detected by electrophoretic or chromatographic methods.
- a selectable marker gene such as the NOS-NPT II-NOS chimeric gene
- a scorable marker gene such as the NOS gene
- any desired gene may be inserted into pMON120 or other which are designed to form co-integrates with plant tumor inducing or similar plasmids.
- pMON120 or other which are designed to form co-integrates with plant tumor inducing or similar plasmids.
- the Applicants have created a variety of chimeric genes, which are discussed in the previously-cited application, “Chimeric Genes Suitable for Expression in Plant Cells.”
- any gene for use in this invention may be determined through routine experimentation by those skilled in the art. Such usage is not limited to chimeric genes; for example, this invention may be used to insert multiple copies of a natural gene into plant cells.
- This invention is suitable for use with a wide variety of plants, as may be determined through routine experimentation by those skilled in the art.
- this invention is likely to be useful to transform cells from any type of plant which can be infected by bacteria from the genus Agrobacterium . It is believed that virtually all dicotyledonous plants, and certain monocots, can be infected by one or more strains of Agrobacterium .
- microorganisms of the genus Rhizobia are likely to be useful for carrying co-integrate plasmids of this invention, as may be determined by those skilled in the art. Such bacteria might be preferred for certain types of transformations or plant types.
- Certain types of plant cells can be cultured in vitro and regenerated into differentiated plants using techniques known to those skilled in the art. Such plant types include potatoes, tomatoes, carrots, alfalfa and sunflowers. Research in in vitro plant culture techniques is progressing rapidly, and methods are likely to be developed to permit the regeneration of a much wider range of plants from cells cultured in vitro. Cells from any such plant with regenerative capacity are likely to be transformable by the in vitro co-cultivation method discussed previously, as may be determined through routine experimentation by those skilled in the art. Such transformed plant cells may be regenerated into differentiated plants using the procedures described in the examples.
- the in vitro co-cultivation method offers certain advantages in the transformation of plants which are susceptible to in vitro culturing and regeneration.
- this invention is not limited to in vitro cell culture methods.
- a variety of plant shoots and cuttings including soybeans, carrots, and sunflowers
- A. tumefaciens cells carrying the co-integrate plasmids of this invention have been transformed by contact with A. tumefaciens cells carrying the co-integrate plasmids of this invention. It is also possible to regenerate virtually any type of plant from a cutting or shoot.
- co-cultivation be utilized to transfer the co-integrate plasmids of this invention into plant cells.
- a variety of other methods are being used to insert DNA into cells. Such methods include encapsulation of DNA in liposomes, complexing the DNA with chemicals such as polycationic substances or calcium phosphate, fusion of bacterial spheroplasts with plant protoplasts, microinjection of DNA into a cell, and induction of DNA uptake by means of electric current.
- This invention may be useful for a wide variety of purposes.
- certain bacterial enzymes such as 5-enol pyruvyl shikimate-3-phosphoric acid synthase (EPSP synthase) are inactivated by certain herbicides; other enzymes, such as glutathione-S-transferase (GST), inactivate certain herbicides.
- GST glutathione-S-transferase
- chimeric genes may be inserted into plants which will cause the plants to create mammalian polypeptides, such as insulin, interferon, growth hormone, etc.
- the plants or cultured plant tissue
- the desired protein may be extracted from the harvested plant tissue.
- An alternate use of this invention is to create plants with high content of desired substances, such as storage proteins or other proteins.
- a plant might contain one or more copies of a gene which codes for a desirable protein. Additional copies of this gene may be inserted into the plant by means of this invention.
- the structural sequence of the gene might be inserted into a chimeric gene under the control of a different promoter which causes prolific transcription of the structural sequence.
- the methods of this invention may be used to identify, isolate, and study DNA sequences to determine whether they are capable of promoting or otherwise regulating the expression of genes within plant cells.
- the DNA from any type of cell can be fragmented, using partial endonuclease digestion or other methods.
- the DNA fragments can be inserted randomly into plasmids similar to pMON128.
- These plasmids instead of having a full chimeric gene such as NOS-NPT II-NOS, will have a partial chimeric gene, with a cleavage site for the insertion of DNA fragments, rather than a NOS promoter or other promoter.
- the plasmids with inserted DNA are then inserted into A.
- tumefaciens where they can recombine with the Ti plasmids.
- Cells having co-integrate plasmids are selected by means of the spc/str or other marker gene.
- the co-integrate plasmids are then inserted into the plant cells, by bacterial co-cultivation or other means.
- the plant cells will contain a selectable marker structural sequence such as the NPT II structural sequence, but this structural sequence will not be transcribed unless the inserted DNA fragment serves as a promoter for the structural sequence.
- the plant cells may be selected by growing them on medium containing kanamycin or other appropriate antibiotics.
- a piece of DNA includes plasmids, phages, DNA fragments, and polynucleotides, whether natural or synthetic.
- a “chimeric piece of DNA” is limited to a piece of DNA which contains at least two portions (i.e., two nucleotide sequences) that were derived from different and distinct pieces of DNA.
- a chimeric piece of DNA cannot be created by merely deleting one or more portions of a naturally existing plasmid.
- a chimeric piece of DNA may be produced by a variety of methods, such as ligating two fragments from different plasmids together, or by synthesizing a polynucleotide wherein the sequence of bases was determined by analysis of the base sequences of two different plasmids.
- a chimeric piece of DNA is limited to DNA which has been assembled, synthesized, or otherwise produced as a result of man-made efforts, and any piece of DNA which is replicated or otherwise derived therefrom.
- Man-made efforts include enzymatic, cellular, and other biological processes, if such processes occur under conditions which are caused, enhanced, or controlled by human effort or intervention; this excluses plasmids, phages, and polynucleotides which are created solely by natural processes.
- the term “derived from” shall be construed broadly. Whenever used in a claim, the term “chimeric” shall be a material limitation.
- a “marker gene” is a gene which confers a phenotypically identifiable trait upon the host cell which allows transformed host cells to be distinguished from non-transformed cells. This includes screenable, scorable, and selectable markers.
- a “region of homology” refers to a sequence of bases in one plasmid which has sufficient correlation with a sequence of bases in a different plasmid to cause recombination of the plasmid to occur at a statistically determinable frequency. Preferably, such recombination should occur at a frequency which allows for the convenient selection of cells having combined plasmids, e.g., greater than 1 per 10 6 cells. This term is described more fully in a variety of publications, e.g., Leemans et al, 1981.
- chimeric gene refers to a gene that contains at least two portions that were derived from different and distinct genes. As used herein, this term is limited to genes which have been assembled, synthesized, or otherwise produced as a result of man-made efforts, and any genes which are replicated or otherwise derived therefrom. “Man-made efforts” include enzymatic, cellular, and other biological processes, if such processes occur under conditions which are caused, enhanced, or controlled by human effort or intervention; this excludes genes which are created solely by natural processes.
- a “gene” is limited to a segment of DNA which is normally regarded as a gene by those skilled in the art.
- a plasmid might contain a plant-derived promoter region and a heterologous structural sequence, but unless those two segments are positioned with respect to each other in the plasmid such that the promoter region causes the transcription of the structural sequence, then those two segments would not be regarded as included in the same gene.
- This invention relates to chimeric genes which have structural sequences that are “heterologous” with respect to their promoter regions. This includes at least two types of chimeric genes:
- DNA of a gene which is foreign to a plant cell For example, if a structural sequence which codes for mammalian protein or bacterial protein is coupled to a plant promoter region, such a gene would be regarded as heterologous.
- a plant cell gene which is naturally promoted by a different plant promoter region For example, if a structural sequence which codes for a plant protein is normally controlled by a low-quantity promoter, the structural sequence may be coupled with a prolific promoter. This might cause a higher quantity of transcription of the structural sequence, thereby leading to plants with higher protein content. Such a structural sequence would be regarded as heterologous with regard to the prolific promoter.
- a chimeric gene of this invention may be created which would be translated into a “fusion protein”, i.e., a protein comprising polypeptide portions derived from two separate structural sequences. This may be accomplished by inserting all or part of a heterologous structural sequence into the structural sequence of a plant gene, somewhere after the start codon of the plant structural sequence.
- a promoter region derived from a specified gene shall include a promoter region if one or more parts of the promoter region were derived from the specified gene. For example, it might be discovered that one or more portions of a particular plant-derived promoter region (such as intervening region 8 , shown on FIG. 12 ) might be replaced by one or more sequences derived from a different gene, such as the gene that contains the heterologous structural sequence, without reducing the expression of the resulting chimeric gene in a particular type of host cell.
- Such a chimeric gene would contain a plant-derived association region 2 , intervening region 4 , and transcription initiation sequence 6 , followed by heterologous intervening region 8 , 5′ non-translated region 10 and structural sequence 14 .
- Such a chimeric gene is within the scope of this invention.
- derived from shall be construed broadly.
- a structural sequence may be “derived from” a particular gene by a variety of processes, including the following:
- the gene may be reproduced by various means such as inserting it into a plasmid and replicating the plasmid by cell culturing, in vitro replication, or other methods, and the desired sequence may be obtained from the DNA copies by various means such as endonuclease digestion;
- mRNA which was coded for by the gene may be obtained and processed in various ways, such as preparing complementary DNA from the mRNA and then digesting the cDNA with endonucleases;
- sequence of bases in the structural sequence may be determined by various methods, such as endonuclease mapping or the Maxam-Gilbert method.
- a strand of DNA which duplicates or approximates the desired sequence may be created by various methods, such as chemical synthesis or ligation of oligonucleotide fragments.
- a structural sequence of bases may be deduced by applying the genetic code to the sequence of amino acid residues in a polypeptide.
- DNA structural sequences may be determined for any polypeptide, because of the redundancy of the genetic code. From this variety, a desired sequence of bases may be selected, and a strand of DNA having the selected sequence may be created.
- any DNA sequence may be modified by substituting certain bases for the existing bases. Such modifications may be performed for a variety of reasons. For example, one or more bases in a sequence may be replaced by other bases in order to create or delete a cleavage site for a particular endonuclease. As another example, one or more bases in a sequence may be replaced in order to reduce the occurrence of “stem and loop” structures in messenger RNA. Such modified sequences are within the scope of this invention.
- a structural sequence may contain introns and exons; such a structural sequence may be derived from DNA, or from an mRNA primary transcript. Alternately, a structural sequence may be derived from processed mRNA, from which one or more introns have been deleted.
- the Applicants have deposited two cultures of E. coli cells containing plasmids pMON128 and pMON154 with the American Type Culture Collection (ATCC). These cells have been assigned ATCC accession numbers 39264 and 39265, respectively.
- the Applicants have deposited two cultures of E. coli cells containing plasmids pMON120 and pMON128 and a culture of A. tumefaciens containing a pMON128::Ti cointegrate plasmid, with the American Type Culture Collection (ATCC). These cells have been assigned ATCC accession numbers 39263, 39264, and 39266, respectively.
- ATCC accession numbers 39263, 39264, and 39266 ATCC accession numbers 39263, 39264, and 39266, respectively.
- the Applicants have claimed cultures of microorganisms having the “relevant characteristics” of these cultures. As used herein, the “relevant characteristics” of a cell culture are limited to those characteristics which make the culture suitable for a use which is disclosed, suggested or made possible by the information contained herein.
- the cells may be made resistant to a particular antibiotic by insertion of a particular plasmid or gene into the cells, or the specified plasmids might be removed from the designated cells and inserted into a different strains of cells.
- Such variations are within the scope of this invention, even though they may amount to improvements in the culture, which undoubtedly will occur after more researchers gain access to these cell cultures.
- the 3.4 kb HindIII-23 fragment was purified by adsorption on glass beads (Vogelstein and Gillespie, 1979) after separation from the other HindIII fragments by electrophoresis on a 0.8% agarose gel.
- the purified 3.4 kb HindIII fragment (1.0 ug) was mixed with 1.0 ug of plasmid pBR327 DNA (Soberon, et al, 1980) that had been digested with both HindIII (2 units, 1 hour, 37° C.) and calf alkaline phosphatase (CAP; 0.2 units, 1 hour, 37° C.), de-proteinized with phenol, ethanol precipitated, and resuspended in 10 ul of TE (10 mM Tris HCl, pH8, 1 mM EDTA).
- T4 DNA ligase prepared by the method of Murray et al, 1979 was added to the fragment mixture.
- One unit is defined as the concentration sufficient to obtain greater than 90% circularization of one microgram of HindIII linearized pBR327 plasmid in 5 minutes at 22° C.
- the mixed fragments were contained in a total volume of 15 ul of 25 mM Tris-HCl pH8, 10 mM MgCl 2 , 1 mM dithiotheitol, 200 uM spermidine HCl and 0.75 mM ATP (ligase buffer).
- the mixture was incubated at 22° for 3 hours and then mixed with E. coli C600 recA56 cells that were prepared for transformation by treatment with CaCl 2 (Maniatis et al, 1982). Following a period for expression of the ampicillin resistance determinant carried by the pBR327 vector, cells were spread on LB solid medium plates (Miller, 1972) containing ampicillin at 200 ug/ml. After incubation at 37° C. for 16 hours, several hundred clones were obtained.
- Plasmid mini-preps (Ish-Horowicz and Burke, 1981) were performed on 24 of these colonies and aliquots of the plasmid DNA's obtained (0.1 ug) were digested with HindIII to demonstrate the presence of the 3.4 kb HindIII fragment.
- One plasmid demonstrated the expected structure and was designated pMON38.
- pMON38 DNA were prepared by Triton X-100 lysis and CsC1 gradient procedure (Davis et al, 1980).
- pMON38 DNA Fifty ug of pMON38 DNA were digested with HindIII and BamHI (50 units each, 2 hours, 37°) and the 2.3 kb HindIII-BamHI fragment was purified as described above. The purified fragment (1 ug) was mixed with 1 ug of the 2.9 kb HindIII-BamHI fragment of the pBR327 vector purified as described above. Following ligation (T4 DNA ligase, 2 units) and transformation of E. coli cells as described above, fifty ampicillin-resistant colonies were obtained. DNAs from twelve plasmid mini-preps were digested with HindIII and BamHI to ascertain the presence of the 2.3 kb fragment. One plasmid of the correct structure was chosen and designated pMON41, as shown in FIG. 2 . A quantity of this DNA was prepared as described above.
- plasmid pMON38 (described in Example 1) were digested with RsaI (30 units, 2 hours, 37° C.) and the 1100 bp RsaI fragment was purified after separation by agarose gel electrophoresis using the glass bead method described in the previous example.
- This DNA was mixed with 0.2 ug of phage M13 mp8RF DNA which had been previously digested with SmaI and BamHI (2 units each, 1 hour, 37°) and 0.2 units of calf alkaline phosphotase (CAP).
- CAP calf alkaline phosphotase
- plasmid pGV3106 Twenty ug of plasmid pGV3106 (Hernalsteens et al 1980, prepared by the method of Currier and Nester 1976) was digested with HindIII (20 units, 2 hours, 37°) and mixed with 2 ug of HindIII-digested pBR327. Following ligation (T4 DNA ligase, 2 units) and transformation of E. coli cells as described above, one colony resistant to trimethoprim (100 ug/ml) and ampicillin was obtained. Digestion of plasmid DNA from this cell demonstrated the presence of a 6 kb HindIII fragment. This plasmid was designated pMON31.
- Plasmid pMON31 from a mini-prep (0.5 ug) was digested with EcoRI (1 unit, 1 hour, 37° C.) and the endonuclease was inactivated by heating (10 min, 70° C.). The 8.5 kb plasmid fragment was re-circularized in a ligation reaction of 100 ul (T4 DNA ligase, 1 unit) and used to transform E. coli cells with selection for ampicillin and streptomycin (25 ug/ml) resistant colonies. Plasmid mini-prep DNA's from six clones were digested with EcoRI to ascertain loss of the 850 by fragment. One plasmid lacking the 850 bp EcoRI fragment was designated pMON53. This plasmid was introduced into E. coli GM42 dam ⁇ cells (Bale et al, 1979) by transformation as described.
- Plasmid pMON53 (0.5 ug) from a mini-prep prepared from dam cells was digested with ClaI, and recircularized in dilute solution as described above. Following transformation of E. coli GM42 cells and selection for ampicillin and spectinomycin (50 ug/ml) resistant clones, fifty colonies were obtained. Digestion of plasmid mini-prep DNA's from six colonies showed that all lacked the 2 kb ClaI fragment. One of these plasmids was designated pMON54, as represented in FIG. 4 . Plasmid DNA was prepared as described in Example 1.
- Plasmid pMON54 DNA (20 ug) was digested with EcoRI and PstI (20 units of each, 2 hours, 37° C.) and the 5.7 kb fragment was purified from agarose gels using NA-45 membrane (Schleicher and Schuell, Keene, N. H.).
- the purified 5.7 kb fragment (0.5 ug) was mixed with 0.3 ug of a 740 bp EcoRI-PstI fragment obtained from M13 mp8 M-4 RF DNA (described in Example 2) which was purified using NA-45 membrane. Following ligation (T4 DNA ligase, 2 units), transformation of E. coli GM42 dam ⁇ cells, and selection for spectinomycin resistant cells, twenty colonies were obtained. Plasmid mini-prep DNA's prepared from twelve of these clones were digested with PstI and EcoRI to demonstrate the presence of the 740 bp fragment. One plasmid carrying this fragment was designated pMON64. quantity of this plasmid DNA was prepared as described in Example 1.
- DNA (0.5 ug) of pMON64 was digested with ClaI (1 unit, 1 hour, 37° C.), the ClaI was heat inactivated, and the fragments rejoined with T4 DNA ligase (1 unit). Following transformation and selection for spectinomycin resistant cells, plasmid mini-preps from twelve colonies were made. The DNA's were digested with BamHI and EcoRI to determine the orientation of the 2 kb ClaI fragment. Half of the clones contained the ClaI fragment in the inverse orientation of that in pMON64. One of these plasmids was designated pMON109, as represented in FIG. 5 . DNA was prepared as described in Example 1.
- Plasmid pNW31C-8,29C (Thomashow et al, 1980) was obtained from Dr. S. Gelvin of Purdue University, West Lafayette, I N. This plasmid carries the pTiA6 7.5 kb Bam-8 fragment. The Bam-8 fragment was purified from 50 ug of BamHI-digested pNW31C-8,29C using NA-45 membrane as described in previous examples.
- the purified 7.5 kb Bam-8 fragment (1.0 ug) was mixed with 0.5 ug of pBR327 vector DNA which had been previously digested with both BamHI (2 units) and 0.2 units of calf alkaline phosphatase (CAP) for 1 hour at 37°; the mixture was deproteinized and resuspended as described in previous examples.
- the mixed fragments were treated with T4 ligase (2 units), used to transform E. coli C600 recA cells and ampicillin-resistant colonies were selected as described previously. Mini-preps to obtain plasmid DNA were performed on twelve of these clones.
- the DNA was digested with BamHI to demonstrate the presence of the pBR327 vector and 7.5 kb Bam-8 fragments. One plasmid demonstrating both fragments was designated pMON90.
- DNA was prepared as described in Example 1.
- pMON90 DNA Twenty-five ug of pMON90 DNA were digested with BglII (25 units, 2 hours, 37°) and the 2.6 kb BglII fragment was purified using NA-45 membrane. To create blunt ends, the fragment (2 ug) was resuspended in 10 ul of 50 mM NaCl, 6.6 mM Tris-HCl pH8, 6.6 mM MgCl 2 and 0.5 mM dithiothreitol (Klenow Buffer). The 4 deoxy-nucleoside triphosphates (dATP, dTTP, dCTP, and dGTP) were added to a final concentration of 1 mM and one unit of E.
- dATP deoxy-nucleoside triphosphates
- Plasmid mini-preps were prepared from twelve colonies and digested with HindIII to determine the size of the recombinant plasmid and with Sinai to determine that the correct fragment had been inserted.
- One plasmid with the correct structure was designated pMON113, as shown in FIG. 6 .
- Plasmid DNA was prepared as described in Example 1.
- plasmid pMON109 (described in Example 3) were digested with EcoRI and BamHI (20 units each, 2 hours, 37° C.) and the 3.4 kb BamHI-EcoRI fragment was purified using NA-45 membrane as described in previous examples.
- Twenty ug of plasmid pMON41 (described in Example 1) were digested with BamHI and PvuI (20 units each, 2 hours, 37° C.) and the 1.5 kb BamHI-PvuI fragment purified using NA-45 membrane as described in previous examples.
- pMON113 DNA Twenty ug of pMON113 DNA (described in Example 4) were digested with PvuI and EcoRI (2 units each, 2 hr, 37° C.) and the 3.1 kb PvuI-EcoRI fragment was purified using NA-45 membrane as above.
- the 3.1 kb EcoRI-PvuI pMON113 fragment 1.5 ug was mixed with 1.5 ug of the 3.4 kb EcoRI-BamHI fragment from pMON109.
- T4 ligase 3 units
- the ligase was inactivated by heating (10 minutes, 70° C.), and 5 units of BamHI was added.
- a culture of E. coli containing pMON120 has been deposited with the American Type Culture Collection. This culture has been assigned accession number 39263.
- Plasmid pMON75 (described in detail in a separate application entitled “Chimeric Genes Suitable for Expression in Plant Cells,” previously cited and incorporated contains a chimeric NOS-NPT II-NOS gene. This plasmid (and pMON128, described below) may be digested by EcoRI and a 1.5 kb fragment may be purified which contains the NOS-NPT II-NOS gene.
- Plasmid pMON120 was digested with EcoRI and treated with calf alkaline phosphatase. After phenol deproteinization and ethanol precipitation, the EcoRI-cleaved pMON120 linear DNA was mixed with 0.5 ug of the 1.5 kb EcoRI chimeric gene fragment from pMON75 or 76. The mixture was treated with 2 units of T4 DNA ligase for 1 hour at 22°. After transformation of E. coli cells and selection of colonies resistant to spectinomycin (50 ug/ml), several thousand colonies appeared. Six of these were picked, grown, and plasmid mini-preps made.
- the plasmid DNA's were digested with EcoRI to check for the 1.5 kb chimeric gene insert and with BamHI to determine the orientation of the insert. BamHI digestion showned that in pMON128 the chimeric gene was transcribed in the same direction as the intact nopaline synthase gene of pMON120.
- a culture of E. coli containing pMON128 has been deposited with the American Type Culture Collection. This culture has been assigned accession number 39264.
- the mixture of cells was cultured in Luria Broth (LB), spread on an LB plate, and incubated for 16 to 24 hours at 30° C. to allow plasmid transfer and generation of co-integrate plasmids.
- the cells were resuspended in 3 ml of 10 mM MgSO 4 and 0.2 ml aliquot was then spread on an LB plate containing 25 ug/ml chloramphenicol and 100 ug/ml each of spectinomycin and streptomycin. After incubation for 48 hr at 30°, approximately 10 colonies were obtained. One colony was chosen and grow at 30° C. in LB medium containing chloramphenicol, spectinomycin, and streptomycin at the concentrations given above.
- a separate type of co-integrate plasmid for use in control experiments was prepared by inserting pMON120 into A. tumefaciens cells, and selecting for cells with co-integrate plasmids using spectinomycin and streptomycin, as described above. Like pMON120, these plasmids do not contain the chimeric NOS-NPT II-NOS gene.
- Mitchell petunia plants were grown in growth chambers with two or three banks of fluorescent lamps and two banks of incandescent bulbs (about 5,000 lux). The temperature was maintained at a constant 21° C. and the lights were on for 12 hours per day. Plants were grown in a 50/50 mix of Vermiculite and Pro-mix BX (Premier Brands Inc., Canada). Plants were watered once a day with Hoagland's nutrient solution. Tissue was taken from dark green plants with compact, bushy growth. Leaves were sterilized in a solution of 10% commercial bleach and a small amount of detergent or Tween 20 for 20 minutes with occasional agitation.
- Leaves were rinsed two or three times with sterile distilled water, Thin strips (about 1 mm) were cut from the leaves, perpendicular to the main rib. The strips were placed in the enzyme mix at a ratio of about 1 g tissue to 10 ml enzymes. The dishes were sealed with parafilm, and incubated in the dark or under low, indirect light while gently agitating continuously (e.g., 40 rpm on gyrotary shaker). Enzymic incubations generally were run overnight, about 16-20 hours.
- the digestion mixture was sieved through a 68, 74, or 88 um screen to remove large debris and leaf material.
- the filtrate was spun at 70-100 g for five minutes to pellet the protoplasts.
- the supernatant was decanted and the pellet was gently resuspended in float rinse solution. This suspension was poured into babcock bottles. The bottles were filled to 2 or 3 cm above the base of the neck. 1 ml of growth medium MS9 was carefully layered on top of the float rinse.
- the Babcock bottles were balanced and centrifuged at 500 to 1000 rpm for 10 to 20 minutes.
- the protoplasts formed a compact band in the neck at the interface.
- the band was removed with a pipette, taking care not to pick up any excess float rinse.
- the protoplasts were diluted into MS9. At this point, the protoplasts were washed with MS9 or diluted for plating without washing.
- Protoplasts were suspended in MS9 medium at 5 ⁇ 10 4 per ml, and plated into T-75 flasks, at 6 ml per flask. Flasks were incubated on a level surface with dim, indirect light or in the dark at 26-28° C.
- MSO medium which does not contain mannitol
- MSO was added to each flask, using an amount equal to one-half the original volume. The same amount of MSO is added again on day 4. This reduces the mannitol concentration to about 0.33 M after the first dilution, and about 0.25 M after the second dilution.
- TXD cells tobacco suspension cultures
- MS2C medium was obtained by mixing 0.8% agar with MS-ES medium, autoclaving the mixture, and cooling the mixture until it solidifies in the plate.
- One ml of the TXD suspension was spread over 25 ml of feeder plate medium.
- An 8.5 cm disc of Whatman #1 filter paper was laid over the TXD feeder cells and smoothed out. A 7 cm disc of the same paper was placed in the center of the larger one.
- aliquots of a culture of A. tumefaciens cells were added to the flasks which contained the plant cells.
- One set of aliquots contained cells with the pMON128::Ti co-integrate plasmids having chimeric NOS-NPT II-NOS genes.
- the other set of aliquots contained cells with the pMON120::Ti co-integrate plasmids, which do not have chimeric NOS-NPT II-NOS genes.
- the bacteria were added to the flasks to a density of 10 8 cells/ml. 0.5 ml of the cell mixture was spread in a thin layer on the surface of the 7 cm filter paper disc. The plates were wrapped in parafilm or plastic bags and incubated under direct fluorescent lighting, no more than five plates in a stack.
- tumefaciens strains containing the pMON120 co-integrate plasmid The kanamycin resistant transformants are capable of sustained growth in culture medium containing kanamycin.
- Southern blotting experiments (as described in E. Southern, J. Mol. Biol. 98: 503 (1975) confirmed that these cells contain the chimeric NOS-NPT II gene.
- the transformed kanamycin-resistant colonies described in Example 10 contained both tumorous and non-tumorous cells, as described in FIG. 9 and the related text. The following procedure was used to isolate non-tumorous transformed cells from tumorous transformed cells, and to regenerate differentiated plant tissue from the non-tumorous cells.
- Colonies were grown on MS104 agar medium containing 30 ug/ml kanamycin sulfate and 500 ug/ml carbenicillin until they reached about 1 cm in diameter. Predominantly tumorous colonies appear a somewhat paler shade of green and are more loosely organized than predominantly non-tumorous colonies. Predominantly non-tumorous colonies were removed from the MS104 medium and placed upon MS11 medium containing 30 ug/ml kanamycin and 500 ug/ml carbenicillin. As the colonies continued to grow, colonies that appeared pale green and loosely organized were removed and discarded.
- MS11 medium contains zeatin, a phytohormone which induces shooting formation in non-tumorous colonies.
- zeatin a phytohormone which induces shooting formation in non-tumorous colonies.
- Several shoots were eventually observed sprouting from kanamycin-resistant colonies. These shoots may be grown to a desired size, cut off by a sharp blade, and inserted into agar medium without phytohormones, such as MSO, where they may generate roots.
- the medium may be supplemented by napthalene acetic acid to induce shooting.
- the shoots may be grown to a desired size in the agar medium, and then transferred into soil. If properly cultivated, such plants will grow to maturity and generate seed.
- the acquired trait will be inherited by progeny according to classic Mendelian genetics.
- Plasmid pBR327 (Soberon et al, 1981), 1 ug, was digested with HindIII and BamHI (2 units each, 2 hours, 37° C.) Following digestion, the endonucleases were heat inactivated and the cleaved pBR327DNA was added to the BamHI-HindIII Tn5 fragments. After addition of ATP to a concentration of 0.75 mM, 10 units of T4 DNA ligase (prepared by the method of Murray et al, 1979) was added, and the reaction was allowed to continue for 16 hours at 12°-14° C. One unit of T4 DNA ligase will give 90% circularization of one ug of HindIII-cleaved pBR327 plasmid in 5 minutes at 22° C.
- the ligated DNA was used to transform CaCl 2 -shocked E. coli C600 recA56 cells (Maniatis et al, 1982). After expression in Luria broth (LB) for 1 hour at 37° the cells were spread on solid LB media plates containing 200 ug/ml ampicillin and 40 ug/ml kanamycin. Following 16 hours incubation at 37° C., several hundred colonies appeared. Plasmid mini-prep DNA was prepared from six of these. (Ish-Horowicz and Burke, 1981). Endonuclease digestion showed that all six of the plasmids carried the 1.8 kb HindIII-BamHI fragment. One of those isolates was designated as pMON1001 as shown in FIG. 17 .
- oligonucleotide with the following sequence, 5′-TGCAGATTATTTGG-3′, was synthesized (Beaucage and Carruthers, 1981, as modified by Adams et al, 1982). This oligonucleotide contained a 32 P radioactive label, which was added to the 5′ thymidine residue by polynucleotide kinase.
- S1A An M13 mp 7 derivative, designated as S1A, was given to Applicants by M. Bevan and M. D. Chilton, Washington University, St. Louis, Mo. To the best of Applicants' knowledge and belief, the S1A DNA was obtained by the following method. A pTiT37 plasmid was digested with HindIII, and a 3.4 kb fragment was isolated and designated as the HindIII-23 fragment. This fragment was digested with Sau3a, to create a 344 bp fragment with Sau3a ends. This fragment was inserted into double-stranded, replicative form DNA from the M13 mp 7 phage vector (Messing et al, 1981) which had been cut with BamHI.
- the mixture was incubated (1 hour, 55° C.), the HaeIII was inactivated (70° C., 3 minutes), and the four dNTP's (1 mM, 12 ul) and T4 DNA polymerase (50 units) were added.
- the mixture was incubated (1 hour, 37° C.) and the polymerase was inactivated (70° C., 3 minutes). This yielded a fragment of about 570 bp.
- EcoRI 150 units was added, the mixture was incubated (1 hour, 37° C.) and the EcoRI was inactivated (70° C., 3 minutes).
- plasmid pMON40 Five ug of plasmid pMON40 (described in Example 13) were digested with BglII (10 units, 1.5 hour, 37° C.), and the BglII was inactivated (70° C., 10 minutes). The four dNTP's (1 mM, 5 ul) and Klenow polymerase (8 units) were added, the mixture was incubated (37° C., 40 minutes), and the polymerase was inactivated (70° C., 10 minutes). EcoRI (10 units) was added and incubated (1 hour, 37° C.), and calf alkaline phosphatase (CAP) was added and incubated (1 hour, 37° C.).
- a fragment of about 3.9 kb was purified on agarose gel using NA-45 membrane (Scheicher and Scheull, Keene N H). The fragment (1.0 pM) was mixed with the NOS promoter fragment (0.1 pM), described in Example 3, and with T4 DNA ligase (100 units). The mixture was incubated (4° C., 16 hours). The resulting plasmids were inserted into E. coli cells, which were selected on media containing 200 ug/ml ampicillin. Thirty-six clonal Amp R colonies were selected, and mini-preps of plasmids were made from those colonies.
- the plasmid from one colony demonstrated a 308 by EcoRI-BglII fragment, a new SstII cleavage site carried by the 308 bp NOS fragment, and a new PstI site.
- This plasmid was designated as pMON58, as shown in FIG. 18 .
- pMON58 DNA was prepared as described above.
- plasmid pMON42 (described in Example 16) prepared from dam— E. coli cells were digested with RsaI and BamHI (50 units of each, 3 hours, 37° C.) and the 720 bp RsaI-BamHI fragment was purified using NA-45 membrane. Eight ug of the purified 720 bp BamHI-RsaI fragment were digested with MboI (10 minutes, 70° C.), the ends were made blunt by filling in with the large Klenow fragment of DNA polymerase I and the-four dNTP's.
- 0.1 ug of the resulting DNA mixture was added to 0.05 ug of M13 mp 8 previously digested with SmaI (1 unit, 1 hour 37° C.) and calf alkaline phosphatase (0.2 units). After ligation (10 units of T4 DNA ligase, 16 hours, 12° C.) and transfection of E. coli JM101 cells, several hundred recombinant phage were obtained. Duplex RF DNA was prepared from twelve recombinant, phage-carrying clones.
- the RF DNA (0.1 ug) was cleaved with EcoRI, (1 unit, 1 hour, 37° C.), end-labeled with 32 P-dATP and Klenow polymerase, and re-digested with BamHI (1 unit, 1 hour, 37° C.).
- EcoRI and BainHI sites span the SmaI site. Therefore, clones containing the 260 bp MboI fragment could be identified as yielding a labelled 270 bp fragment after electrophoresis on 6% poly-acrylamide gels and autoradiography. Four of the twelve clones carried this fragment.
- the orientation of the insert was determined by digestion of the EcoRI-cleaved, end-labeled RF DNA (0.1 ug) with HinfI (1 unit, 1 hour, 37° C.). HinfI cleaves the 260 by MboI fragment once 99 bp from the 3′ end of the fragment and again 42 bp from the end nearest the NOS coding region. Two clones of each orientation were obtained. One clone, digested as M-2 as shown in FIG. 19 , contained the 260 bp fragment with the EcoRI site at the 3′ end of the fragment. M-2 RF DNA was prepared using the procedures of Messing, et al 1981.
- Fifty ug of M-2 RF DNA (described in Example 17) were digested with 50 units of EcoRI and 50 units of BamHI for 2 hours at 37°.
- the 270 bp fragment (1 ug) was purified using agarose gel and NA-45 membrane.
- Plasmid pMON58 (described in Example 4) was digested with EcoRI and BamHI (50 ug, 50 units each, 2 hours, 37° C.) and the 1300 bp fragment was purified using NA-45 membrane.
- the 270 bp EcoRI-BamHI (0.1 ug) and 1300 bp EcoRI-BamHI (0.5 ug) fragments were mixed, treated with T4 DNA ligase (2 units) for 12 hours at 14° C.
- Plasmid pMON38 is a clone of the pTiT37 HindIII-23 fragment inserted in the HindIII site of pBR327 (Soberon, et al 1980).
- pMON38 DNA (20 ug) was digested with EcoRI (20 units, 2 hours, 37° C.) and calf alkaline phosphatase (0.2 units, 1 hour, 37° C.) The pMON38 DNA reaction was extracted with phenol, precipitated with ethanol, dried and resuspended in 20 ul of 10 mM Tris-HCl, 1 mM EDTA, pH 8.
- 0.2 ug of the cleaved pMON38 DNA was added to the chimeric gene mixture described above.
- the mixture was treated with T4 DNA ligase (4 units, 1 hour, 22° C.) and mixed with Rb chloride-treated E. coli C600 recA56 cells to obtain transformation. After plating with selection for ampicillin-resistant (200 ug/ml) colonies, 63 potential candidates were obtained.
- Alkaline mini-preps of plasmid DNA were made from 12 of these and screened by restriction endonuclease digestion for the proper constructs. Plasmid DNA's that contained a 1.5 kb EcoRI fragment and a new BgII site were digested with BamHI to determine the orientation of the 1.5 kb EcoRI fragment. One of each insert orientation was picked.
- One plasmid was designated pMON75 and the other pMON76, as shown in FIG. 20 . DNA from these plasmids were prepared as described in previous examples.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
5′-GTAC
CATG
5′ . . . CTGCA
. . . GACGT
and one cohesive end (at the EcoRI site) with a sequence of The shorter strand was about 308 bp long.
5′CCGGATCCGG,
GGCCTAGGCC
into the SmaI cleavage site. This eliminated the SmaI cleavage site and replaced it with a BamHI cleavage site. A second BamHI cleavage site already existed, about 500 bp from the new BamHI site. The Applicants digested the plasmid with BamHI, separated the 500 bp fragment from the 4.2 kb fragment, and circularized the 4.2 kb fragment. The resulting plasmids were inserted into E. coli, which were then selected for resistance to ampicillin and kanamycin. A clonal colony of E. coli was selected; these cells contained a plasmid which was designated as pMON40, as shown in
-
- The Applicants deleted the NPTII promoter from pMON40, and replaced it with the NOS promoter fragment described previously, by the following method, shown on
FIG. 18 .
- The Applicants deleted the NPTII promoter from pMON40, and replaced it with the NOS promoter fragment described previously, by the following method, shown on
5′ | - AGATC | GATCT- | |
- TCTAG | CTAGA-5′ |
-
- In an alternate preferred embodiment of this invention, a chimeric gene was created comprising (1) a sbss promoter region and 5′ non-translated region, (2) a structural sequence which codes for bovine growth hormone (BGH) and (3) a
NOS 3′ non-translated region. This chimeric gene was created as follows.
- In an alternate preferred embodiment of this invention, a chimeric gene was created comprising (1) a sbss promoter region and 5′ non-translated region, (2) a structural sequence which codes for bovine growth hormone (BGH) and (3) a
per liter | ||||
Enzyme mix: | Cellulysin | 5 | g | ||
Macerozyme | 0.7 | g | |||
Ampicillin | 0.4 | g | |||
KH2PO4 | 27.2 | mg | |||
KNO3 | 101 | mg | |||
CaCl2 | 1.48 | g | |||
MgSO4•7H2O | 246 | mg | |||
KI | 0.16 | mg | |||
CuSO4•5H2O | 0.025 | mg | |||
Mannitol | 110 | g | |||
MS9: | MS salts(see below) | 4.3 | g | ||
Sucrose | 30.0 | g | |||
B5 vitamins (see below) | 1 | ml | |||
Mannitol | 90.0 | g | |||
Phytohormones: | |||||
Benzyladenine (BA) | 0.5 | | |||
2,4- | 1 | mg | |||
MS-ES | MS salts | 4.3 | g | ||
Sucrose | 30 | | |||
B5 Vitamins | |||||
1 | ml | ||||
Mannitol | 30 | | |||
Carbenicillin | |||||
10 | mg | ||||
Phytohormones: | |||||
Indole acetic acid | 0.1 | mg | |||
MSO: | MS salts | 4.3 | g | ||
Sucrose | 30.0 | | |||
B5 vitamins | |||||
1 | ml | ||||
Feeder plate | MS salts | 4.3 | g | ||
medium: | Sucrose | 30.0 | | ||
B5 vitamins | |||||
1 | ml | ||||
Mannitol | 30.0 | g | |||
Phytohormones: | |||||
BA | 0.5 | mg | |||
Ms2C | MS salts | 4.3 | g | ||
Sucrose | 30 | | |||
B5 vitamins | |||||
1 | ml | ||||
Phytohormones: | |||||
chlorophenoxyacetic | 2 | mg | |||
acid | |||||
MS104: | MS salts | 4.3 | g | ||
Sucrose | 30.0 | | |||
B5 vitamins | |||||
1 | ml | ||||
Phytohormones: | |||||
BA | 0.1 | | |||
NAA | |||||
1 | mg | ||||
MS11: | MS salts | 4.3 | g | ||
Sucrose | 30.0 | | |||
B5 vitamins | |||||
1 | ml | ||||
Phytohormones: | |||||
| 1 | mg | |||
B5 Vitamin | myo-inositol | 100 | g | ||
stock: | thiamine HCl | 10 | g | ||
nicotinic acid | 1 | g | |||
pyrodoxine HCl | 1 | g | |||
Float rinse: | MS salts | 0.43 | g | ||
Sucrose | 171.2 | g | |||
PVP-40 | 40.0 | g | |||
MS salts are purchased pre-mixed as a dry powder from Gibco Laboratories, Grand Island, N.Y.
-
- Five ug of plasmid pMON1001 (described in Example 12) was digested with SmaI. The reaction was terminated by phenol extraction, and the DNA was precipitated by ethanol. A BamHI linker CCGGATCCGG (0.1 ug), which had been phosphorylated with ATP and T4 polynucleotide kinase (Bethesda Research Laboratory, Rockville, Md.) was added to 1 ug of the
pMON 1001 fragment. The mixture was treated with T4 DNA ligase (100 units) for 18 hours at 14° C. After heating at 70° C. for 10 minutes to inactivate the DNA ligase, the DNA mixture was digested with BamHI endonuclease (20 units, 3 hours, 37° C.) and separated by electrophoresis on an 0.5% agarose gel. The band corresponding to the 4.2 kb SmaI-BamHI vector fragment was excised from the gel. The 4.2 kb fragment was purified by absorption on glass beads (Vogelstein and Gillespie, 1979), ethanol precipitated and resuspended in 20 ul of DNA ligase buffer with ATP. T4 DNA ligase (20 units) was added and the mixture was incubated for 1.5 hours at room temperature. The DNA was mixed with rubidium chloride-shocked in E. coli C600 cells for DNA transformation. (Maniatis et al, 1982). After expression for 1 hour at 37° C. in LB, the cells were spread on LB plates containing 200 ug/ml of ampicillin and 20 ug/ml kanamycin. The plates were incubated at 37° C. for 16 hours. Twelve ampicillin-resistant, kanamycin-resistant colonies were chosen, 2 ml cultures were grown, and mini-plasmid preparations were performed. Endonuclease mapping of the plasmids revealed that ten of the twelve contained no SmaI site and a single BamHI site, and were of the appropriate size, 4.2 kb. The plasmid from one of the ten colonies was designated as pMON40, as shown inFIG. 17 .
- Five ug of plasmid pMON1001 (described in Example 12) was digested with SmaI. The reaction was terminated by phenol extraction, and the DNA was precipitated by ethanol. A BamHI linker CCGGATCCGG (0.1 ug), which had been phosphorylated with ATP and T4 polynucleotide kinase (Bethesda Research Laboratory, Rockville, Md.) was added to 1 ug of the
-
- Plasmid pBR325-HindIII-23, a derivative of plasmid pBR325 (Bolivar, 1978) carrying the HindIII-23 fragment of pTIT37 (see
FIG. 14 ) in the HindIII site, was given to Applicants by M. Bevan and M. D. Chilton, Washington University, St. Louis, Mo. DNA of this plasmid was prepared and 30 ug were digested with HindIII (50 units) and BamHI (50 units). The 1.1 kb HindIII-BamHI fragment was purified by adsorption on glass beads (Vogelstein and Gillespie, 1979) after agarose gel electrophoresis. The purified fragment (0.5 ug) was added to 0.5 ug of the 2.9 kb HindIII-BamHI fragment of pBR327. After treatment with DNA ligase (20 units, 4 hours, 22° C.), the resulting plasmids were introduced to E. coli C600 cells. Clones resistant to ampicillin at 200 ug/ml were selected on solid media; 220 clones were obtained. Minipreps of plasmid DNA were made from six of these clones and tested with the presence of a 1.1 kb fragment after digestion with HindIII and BamHI. One plasmid which demonstrated the correct insert was designated pMON42. Plasmid pMON42 DNA was prepared as described in previous examples.
- Plasmid pBR325-HindIII-23, a derivative of plasmid pBR325 (Bolivar, 1978) carrying the HindIII-23 fragment of pTIT37 (see
- A. Bale et al, Mut. Res. 59: 157 (1979)
- F. Bolivar, Gene 4: 121 (1978)
- A. Braun and H. Wood, Proc. Natl. Acad. Sci. USA 73: 496 (1976)
- M. D. Chilton et al, Cell 11: 263 (1977)
- A. Colman et al, Eur. J. Biochem 91: 303-310 (1978)
- T. Currier and E. Nester, J. Bact. 126: 157 (1976)
- M. Davey et al, Plant Sci. Lett. 18: 307 (1980)
- R. W. Davis et al, Advanced Bacterial Genetics Cold Spring Harbor Laboratory, New York, (1980)
- H. De Greve et al, Plasmid 6: 235 (1981)
- G. Ditta et al, Proc. Natl. Acad. Sci. USA 77: 7347 (1980)
- D. Garfinkel et al, Cell 27: 143 (1981)
- S. Hasezawa et al, Mol. Gen. Genet. 182: 206 (1981)
- J. Hernalsteens et al, Nature 287: 654 (1980)
- D. Ish-Horowicz and J. F. Burke, Nucleic Acids Res. 9: 2989-2998 (1981)
- B. Koekman et al, J. Bacteriol. 141: 129 (1979)
- F. Krens et al, Nature 296: 72 (1982)
- J. Leemans et al, J. Mol. Appl. Genet. 1: 149 (1981)
- J. Leemans et al, The EMBO J. 1: 147 (1982)
- A. L. Lehninger, Biochemistry, 2nd ed. (Worth Publ., 1975)
- P. Lurquin, Nucleic Acids. Res. 6: 3773 (1979)
- T. Maniatis et al, Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Labs, 1982)
- L. Marton et al, Nature 277: 129 (1979)
- T. Matzke and M-D Chilton, J. Mol. Appl. Genet. 1: 39 (1981)
- J. Messing et al, Nucleic Acids Res. 9: 309 (1981)
- J. Messing and J. Vieira, Gene 19:.269-276 (1982)
- J. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, N.Y. (1972)
- N. Murray et al, J. Mol. Biol. 132: 493 (1979)
- G. Ooms et al, Plasmid 7: 15 (1982)
- L. Otten and R. Schilperoort, Biochim. Biophys Acta 527: 497 (1978)
- L. Otten, Mol. Gen. Genet. 183: 209 (1981)
- R. Roberts, Nucleic Acids Res. 10: r117 (1982)
- A. Rorsch and R. Schilperoort, Genetic Engineering, 189 Elsevier/North Holland, N.Y. (1978)
- J. K. Setlow and A. Hollaender, Genetic Engineering, Principles and Methods (Plenum Press 1979)
- X. Soberon et al, Gene 9: 287 (1980)
- L. Stryer, Biochemistry, 2nd. ed. (W. H. Freeman and Co., 1981)
- M. Thomashow et al, Cell 19: 729 (1980)
- B. Vogelstein and D. Gillespie, Proc. Natl. Acad. Sci.: 615-619 (1979)
- L. Willmitzer et al, Nature 287: 359 (1980)
- L. Willmitzer et al, The EMBO J. 1: 139 (1982)
- N. Yadev et al, Nature 287: 1 (1980)
- F. Yang et al, Mol. Gen. Genet. 177: 707 (1980)
- P. Zambryski et al, J. Mol. Appl. Genet. 1:361 (1982)
- S. Adams et al,
Abstract # 149, 183rd Meeting of the Amer. Chemical Society (1982) - N. Amrhein et al, Plant Physiol. 66: 830 (1980)
- E Auerswald et al, Cold Spr. Hbr. Symp. Quant. Biol. 45: 107 (1981)
- S. Beaucage and M. Carruthers, Tetrahedron Lett. 22: 1859 (1981)
- E. Beck et al, Gene 19: 327 (1982)
- J. Beggs, Nature 275: 104 (1978)
- D. Berg et al, Proc. Natl. Acad. Sci. USA 76: 3628 (1975)
- M. Capecchi, Cell 22: 479 (1980)
- A. C. Y. Chang and S. U. Cohen, J. Bacteriol. 134: 1141-1156 (1978)
- F. Colbece-Garapin, et al, J. Mol. Biol. 150:1-14 (1981)
- Covey, S. N., G. P. Lomonosoff and R. Hull (1981) Nucleic Acids Res., 9:6735-6747
- T. Currier and E. Nester, J. Bact. 126: 157 (1976)
- M. Davey et al, Plant Sci. Lett. 18: 307 (1980)
- Dudley, R. et al., Virology 117:19 (1982)
- R. Fischer and R. Goldberg, Cell 29: 651 (1982)
- R. Fraley and D. Papahadjopoulos, Current Topics in Microbiology and Immunology 96: 171 (1981)
- Fraley, R. T., R. B. Horsch, A. Matzke, M. D. Chilton, W. S. Chilton and P. R. Sanders,
Plant Molecular Biology 3, 371-378 (1984) - Frank, A., H. Guilley, G. Joward, K. Richards and L. Hirth, Cell 21, 285-294 (1980)
- Gardner, R. C. et al., Nucleic Acids Research Vol. 9, No. 12:287 (1981)
- D. Garfinkel et al, Cell 27: 143 (1981)
- L. Guarente, et al, Science 209: 1428-1430 (1980)
- Howarth, A. S. et al., Virology 112:678 (1981)
- J. Hyldig-Nielsen, Nucleic Acids Res. 10: 689 (1982)
- K. Itakura, et al, Science 198: 1056-1063 (1977)
- A. Jimenez and J. Davies, Nature 287: 869 (1980).
- M. Kozak, Cell 15: 1109 (1978)
- S. McKnight, Cell 31: 355 (1982)
- J. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, N.Y. (1972)
- J. Miller and W. Reznikof, The Operon, 2nd edition, Cold Spring Harbor Laboratory, New York (1982)
- N. Murray et al, J. Mol. Biol. 132: 493 (1979)
- H. Pederson et al, Cell 29: 1015 (1982)
- A. Petit and J. Tempe, Mol. Gen. Genet. 167: 145 (1978)
- J. Pittard and B. Wallace, J. Bacteriol. 91: 1494 (1966)
- C. M. Radding, Annu. Rev. Biochem. 47: 847-880 (1978)
- N. Rao and S. Rogers, Gene 7: 79 (1979)
- M. Rassoulzadegan et al, Nature 295: 257 (1982)
- T. Roberts et al, Proc. Natl. Acad. Sci. USA 76: 760 (1979)
- K. Sakaguchi and M. Okanishi, Molecular Breeding and Genetics of Applied Microorganisms, Kodansha/Academic Press (1981)
- D. Sciaky et al, Plasmid 1: 238 (1978)
- D. Shah et al, Proc. Natl. Acad. Sci. USA 79: 1022 (1982)
- T. Shibata et al, Proc. Natl. Acad. Sci. USA 76: 1638-1642 (1979)
- P. Southern and P. Berg, J. Mol. Appl. Gen. 1: 327-341 (1982)
- K. Struhl et al, Proc. Natl. Acad. Sci. USA 75: 1929 (1979)
- L. Stryer, Biochemistry, 2nd edition (Freeman & Co., San Francisco, 1981)
- J. Vieira and J. Messing, Gene 19: 259 (1982)
- T.-K. Wong and E. Neumann, Bioch. Biophys. Res. Comm. 107: 584 (1982)
- R. Woychik et al, Nucleic Acids Res. 10: 7197 (1982)
Claims (21)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US06/783,336 US8334139B1 (en) | 1983-01-17 | 1985-10-04 | Plasmids for transforming plant cells |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US45841183A | 1983-01-17 | 1983-01-17 | |
US45841483A | 1983-01-17 | 1983-01-17 | |
US06/783,336 US8334139B1 (en) | 1983-01-17 | 1985-10-04 | Plasmids for transforming plant cells |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US45841183A Continuation | 1983-01-17 | 1983-01-17 |
Publications (1)
Publication Number | Publication Date |
---|---|
US8334139B1 true US8334139B1 (en) | 2012-12-18 |
Family
ID=47325238
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US06/783,336 Active US8334139B1 (en) | 1983-01-17 | 1985-10-04 | Plasmids for transforming plant cells |
Country Status (1)
Country | Link |
---|---|
US (1) | US8334139B1 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10894812B1 (en) | 2020-09-30 | 2021-01-19 | Alpine Roads, Inc. | Recombinant milk proteins |
US10947552B1 (en) | 2020-09-30 | 2021-03-16 | Alpine Roads, Inc. | Recombinant fusion proteins for producing milk proteins in plants |
US11236347B2 (en) | 2015-08-28 | 2022-02-01 | Pioneer Hi-Bred International, Inc. | Ochrobactrum-mediated transformation of plants |
US11840717B2 (en) | 2020-09-30 | 2023-12-12 | Nobell Foods, Inc. | Host cells comprising a recombinant casein protein and a recombinant kinase protein |
Citations (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4237224A (en) * | 1974-11-04 | 1980-12-02 | Board Of Trustees Of The Leland Stanford Jr. University | Process for producing biologically functional molecular chimeras |
EP0027662A1 (en) | 1979-10-23 | 1981-04-29 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Eucaryotic cells and eucaryotic protoplasts with an amount of naked DNA introduced by lipid vesicles, process for their preparation and their use in the preparation of gene products |
FR2500847A1 (en) | 1981-03-02 | 1982-09-03 | Pasteur Institut | SELECTIVE GENETIC MARKERS FOR EUKARYOTIC CELLS, PROCESS FOR IMPLEMENTING SUCH MARKERS AND APPLICATION OF THE CELLS CONTAINING SUCH A MARKER TO THE MANUFACTURE OF DETERMINED PROTEINS AFTER THEIR TRANSFORMATION BY A CORRESPONDING DNA |
EP0067553A2 (en) | 1981-05-27 | 1982-12-22 | National Research Council Of Canada | An RNA plant virus vector or portion thereof, a method of construction thereof, and a method of producing a gene derived product therefrom |
WO1983001176A1 (en) | 1981-10-01 | 1983-04-14 | Int Plant Research Inst | Process for the genetic modification of cereals with transformation vectors |
EP0086537A1 (en) | 1982-02-11 | 1983-08-24 | Rijksuniversiteit Leiden | A process for the in-vitro transformation of plant protoplasts with plasmid DNA |
US4407956A (en) | 1981-03-13 | 1983-10-04 | The Regents Of The University Of California | Cloned cauliflower mosaic virus DNA as a plant vehicle |
US4459355A (en) | 1982-07-12 | 1984-07-10 | International Paper Company | Method for transforming plant cells |
WO1984002913A1 (en) | 1983-01-17 | 1984-08-02 | Monsanto Co | Chimeric genes suitable for expression in plant cells |
EP0116718A1 (en) | 1983-01-13 | 1984-08-29 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Process for the introduction of expressible genes into plant cell genomes and agrobacterium strains carrying hybrid Ti plasmid vectors useful for this process |
EP0122791A1 (en) | 1983-04-15 | 1984-10-24 | Mycogen Plant Science, Inc. | Plant gene expression |
US4536475A (en) * | 1982-10-05 | 1985-08-20 | Phytogen | Plant vector |
US4652525A (en) * | 1978-04-19 | 1987-03-24 | The Regents Of The University Of California | Recombinant bacterial plasmids containing the coding sequences of insulin genes |
US4762785A (en) | 1982-08-12 | 1988-08-09 | Calgene, Inc. | Novel method and compositions for introducting alien DNA in vivo |
US4886753A (en) | 1986-01-28 | 1989-12-12 | A/S De Danske Sukkerfabrikker | Method for the expression of genes in plants, parts of plants, and plant cell cultures, and DNA fragments, plasmids, and transformed microorganisms to be used when carrying out the method, as well as the use thereof for the expression of genes in plants |
US5034322A (en) * | 1983-01-17 | 1991-07-23 | Monsanto Company | Chimeric genes suitable for expression in plant cells |
EP0142924B1 (en) | 1983-09-26 | 1992-04-15 | Mycogen Plant Science, Inc. | Insect resistant plants |
US5106739A (en) * | 1989-04-18 | 1992-04-21 | Calgene, Inc. | CaMv 355 enhanced mannopine synthase promoter and method for using same |
US5352605A (en) * | 1983-01-17 | 1994-10-04 | Monsanto Company | Chimeric genes for transforming plant cells using viral promoters |
US6051757A (en) | 1983-01-14 | 2000-04-18 | Washington University | Regeneration of plants containing genetically engineered T-DNA |
US6174724B1 (en) | 1983-01-17 | 2001-01-16 | Monsanto Company | Chimeric genes suitable for expression in plant cells |
-
1985
- 1985-10-04 US US06/783,336 patent/US8334139B1/en active Active
Patent Citations (26)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4237224A (en) * | 1974-11-04 | 1980-12-02 | Board Of Trustees Of The Leland Stanford Jr. University | Process for producing biologically functional molecular chimeras |
US4652525A (en) * | 1978-04-19 | 1987-03-24 | The Regents Of The University Of California | Recombinant bacterial plasmids containing the coding sequences of insulin genes |
CA1169793A (en) | 1979-10-23 | 1984-06-26 | Peter H. Hofschneider | Eucaryotic cells, eucaryotic protoplasts and multicellular eucaryotic living organisms containing dna introduced by lipid vesicles, process for their preparation and their use in gene products for immunization and for curing genetically based defects |
EP0027662A1 (en) | 1979-10-23 | 1981-04-29 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Eucaryotic cells and eucaryotic protoplasts with an amount of naked DNA introduced by lipid vesicles, process for their preparation and their use in the preparation of gene products |
FR2500847A1 (en) | 1981-03-02 | 1982-09-03 | Pasteur Institut | SELECTIVE GENETIC MARKERS FOR EUKARYOTIC CELLS, PROCESS FOR IMPLEMENTING SUCH MARKERS AND APPLICATION OF THE CELLS CONTAINING SUCH A MARKER TO THE MANUFACTURE OF DETERMINED PROTEINS AFTER THEIR TRANSFORMATION BY A CORRESPONDING DNA |
WO1982003087A1 (en) | 1981-03-02 | 1982-09-16 | Garapin Florence | Selective genetic markers for eucaryotic cells,method for implementing such markers and application of cells containing such marker to the production of proteins determined after their transformation by a corresponding dna |
US4407956A (en) | 1981-03-13 | 1983-10-04 | The Regents Of The University Of California | Cloned cauliflower mosaic virus DNA as a plant vehicle |
EP0067553A2 (en) | 1981-05-27 | 1982-12-22 | National Research Council Of Canada | An RNA plant virus vector or portion thereof, a method of construction thereof, and a method of producing a gene derived product therefrom |
WO1983001176A1 (en) | 1981-10-01 | 1983-04-14 | Int Plant Research Inst | Process for the genetic modification of cereals with transformation vectors |
EP0086537A1 (en) | 1982-02-11 | 1983-08-24 | Rijksuniversiteit Leiden | A process for the in-vitro transformation of plant protoplasts with plasmid DNA |
US4459355A (en) | 1982-07-12 | 1984-07-10 | International Paper Company | Method for transforming plant cells |
US4762785A (en) | 1982-08-12 | 1988-08-09 | Calgene, Inc. | Novel method and compositions for introducting alien DNA in vivo |
US4536475A (en) * | 1982-10-05 | 1985-08-20 | Phytogen | Plant vector |
EP0116718A1 (en) | 1983-01-13 | 1984-08-29 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Process for the introduction of expressible genes into plant cell genomes and agrobacterium strains carrying hybrid Ti plasmid vectors useful for this process |
EP0290799A2 (en) | 1983-01-13 | 1988-11-17 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Process for the introduction of expressible genes into plant-cell genomes and agrobacterium strains carrying hybrid ti plasmid vectors useful for this process |
US6051757A (en) | 1983-01-14 | 2000-04-18 | Washington University | Regeneration of plants containing genetically engineered T-DNA |
US5352605A (en) * | 1983-01-17 | 1994-10-04 | Monsanto Company | Chimeric genes for transforming plant cells using viral promoters |
EP0131623A1 (en) | 1983-01-17 | 1985-01-23 | Monsanto Co | Chimeric genes suitable for expression in plant cells. |
US6174724B1 (en) | 1983-01-17 | 2001-01-16 | Monsanto Company | Chimeric genes suitable for expression in plant cells |
US5034322A (en) * | 1983-01-17 | 1991-07-23 | Monsanto Company | Chimeric genes suitable for expression in plant cells |
WO1984002913A1 (en) | 1983-01-17 | 1984-08-02 | Monsanto Co | Chimeric genes suitable for expression in plant cells |
US5530196A (en) | 1983-01-17 | 1996-06-25 | Monsanto Company | Chimeric genes for transforming plant cells using viral promoters |
EP0122791A1 (en) | 1983-04-15 | 1984-10-24 | Mycogen Plant Science, Inc. | Plant gene expression |
EP0142924B1 (en) | 1983-09-26 | 1992-04-15 | Mycogen Plant Science, Inc. | Insect resistant plants |
US4886753A (en) | 1986-01-28 | 1989-12-12 | A/S De Danske Sukkerfabrikker | Method for the expression of genes in plants, parts of plants, and plant cell cultures, and DNA fragments, plasmids, and transformed microorganisms to be used when carrying out the method, as well as the use thereof for the expression of genes in plants |
US5106739A (en) * | 1989-04-18 | 1992-04-21 | Calgene, Inc. | CaMv 355 enhanced mannopine synthase promoter and method for using same |
Non-Patent Citations (236)
Title |
---|
A. Kleinhofs and R. Behki, "Prospects for Plant Genome Modification by Nonconventional Methods," Ann. Rev. Genet. 1977 11: 79-101 (1977). |
Abstract by M.D. Chilton et al (p. 14-15), from "Advances in Gene Technology: Molecular Genetics of Plants and Animals" distributed to attendees at the fifteenth Miami Winter Symposium, Jan. 17-21, 1983. (Oral presentation corresponding to patent made on Jan. 18, 1983). |
Abstracts from "Advances in Gene Technology: Molecular Genetics Plants and Animals," 15th Miami Winter Symposium, Jan. 1983. |
Ahmad, F. et al, "From gene to protein: translation into biotechnology" Miami Winter Symposium, vol. 19, 514 (1982). |
Alberts et al, eds. pp. 406-408 In: Molecular Biology of the Cell, Garland Publishing: New York (1983). * |
Ammerer et al, "Recombinant DNA," Proceedings of the Third Cleveland Symposium on Macromolecules, 185-197 (1981). |
Barker et al., "Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens Octopine Ti plasmid pTi15955", Plant Molecular Biology, 2:335-350 (1983). |
Barton et al, "Bacillus thuringiensis δ-Endotoxin Expressed in Transgenic Nicotiana tabacum Provides Resistance to Lepidopteran Insects", Plant Physiol, 85:1103-1109 (1987). |
Barton et al. Cell 32: 1033-1043 (Apr. 1983). * |
Barton et al., "Regeneration of Intact Tobacco Plants Containing Full Length Copies of Genetically Engineered T-DNA, and Transmission of T-DNA to R1 Progeny", Cell, 32:1033-1043 (1983). |
Barton et al., "Tobacco Plants Regenerated from Cells Transformed with an Engineered Ti Plasmid Contain the Gene Encoding Yeast Alcohol Dehydrogenase I", N.A.T.O. FEBS conference, Italy, p. 152, Aug. 23-Sep. 2, 1982. |
Beachy et al., "Accumulation and assembly of soybean mu-coglycimin in seeds of transformed petunia plants", EMBO J., 4(12):3047-3053 (1985). |
Beachy et al., "Accumulation and assembly of soybean μ-coglycimin in seeds of transformed petunia plants", EMBO J., 4(12):3047-3053 (1985). |
Beachy et al., "Molecular characterization of a soybean variety lacking a subunit of the 7S seed storage protein", Plant Molecular Biology, Arco Solar-UCLA Symposium, Apr. 16-22, pp. 413-422 (1983). |
Beck et al, "Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5," Gene 19:327-336 (1982). |
Bedbrook et al., "Molecular cloning and sequencing of cDNA encoding the precursor to the small subunit of chloroplast ribulose-1,5-bisphosphate carboxylase", Nature, 287:692-697 (1980). |
Bennetzen et al. Journal of Biological Chemistry 257(6): 3018-3025 (1982). * |
Beringer et al, "Transfer of the drug resistance transposon Tn 5 to Rhizobium", Nature 276:633-634 (Dec. 7, 1978). |
Berry-Lowe, S. L., "The Nucleotide Sequence . . . Encoding the Small Subunit of Ribulose—1,5-Bisphosphate Carboxylase in Soybean" J. Mol. Appl. Gen. 1:483-498 (1982). |
Bevan et al, "Expression of Bacterial Genes in Higher Plants," J. of Cellular Biology, Supp. 7B, Abstract 1212, p. 268 (1983). |
Bevan et al., "A chimaeric antibiotic resistance gene as a selectable marker for plant cell transformation", Nature, 304:184-187 (1983). |
Bevan et al., "Multiple Transcripts of T-DNA Detected in Nopaline Crown Gall Tumors", Journal of Molecular and Applied Genetics, 1(6):539-546 (1982). |
Bevan et al., "T-DNA of the Agrobacterium Ti and Ri Plasmids", Ann. Rev. Genet., 16:357-384 (1982). |
Bohn et al., "Immunity to Fusarium Wilt in the Tomato", Science, 89(2322):603-604 (1939). |
Brinster et al, "Somatic Expression of Herpes Thymidine Kinase in Mice following Injection of a Fusion Gene into Eggs", Cell 27:223-231 (1981). |
Brisson & Verma, "Soybean leghemoglobin gene family: Normal, pseudo, and truncated genes", Proc. Natl. Acad. Sci. USA, 79:4055-4059 (1982). |
Brisson et al. Proc. Natl. Acad. Sci. USA 79: 4055-4059 (Jul. 1982). * |
Brogile et al., "Light-regulated expression of a pea ribulose-1,5-bisphosphate carboxylase small subunit gene in transformed plant cells", Science, 224:838-843 (1984). |
Brogile et al., "Structural analysis of nuclear genes coding for the precursor to the small subunit of wheat ribulose-1,5-bisphosphate carboxylase", Bio/Technology, 55-61 (1983). |
Cairnes et al., "Translation of animal virus-RNA in the cytoplasm of a plant cell", PNAS, 75(11):5557-5559 (1978). |
Cairns et al., "Expression of a DNA Animal Virus Genome in a Plant Cell", FEBS Letters, 96(2):295-297 (1978). |
Chemical Abstracts of French patent 2500847 1982. * |
Chilton et al, Nature vol. 295 pp. 432-434. * |
Chilton et al, Stadler Symp. vol. 13 pp. 39-52 (1981). * |
Chilton et al., "Agrobacterium rhizogenes inserts T-DNA into the genomes of the host plant root cells", Nature, 295:432-434 (1982). |
Chilton et al., "Cloning Vectors for Plant Genetic Engineering", Plant Genetic Engineering, (1993). |
Chilton et al., "Multiple Viral Specific Transcripts from the Genome of Cauliflower Mosaic Virus", Proceedings of the Miami Winter Symposium, Jan. 1983, published in Advances in Gene Technology: Molecular Genetics of Plants and Animals (Ahmad et al., eds.), p. 564 (1983). |
Chilton et al., "T-DNA from Agrobacterium Ti plasmid in the nuclear DNA fraction of crown gall tumor cells", Proc. Natl. Acad. Sci. USA, 77(7):4060-4064 (1977). |
Chilton et al., "Ti and Ri Plasmids as Vectors for Genetic Engineering of Higher Plants", Proceedings of the Miami Winter Symposium, Jan. 1983, published in Advances in Gene Technology: Molecular Genetics of Plants and Animals (Ahmad et al., eds.), pp. 14-15 (1983). |
Chilton, Genetic Engineering of Osmoregulation, Plenum. Publ. New York pp. 23-39 (1980). * |
Cocking et al., "Aspects of plant genetic manipulation" Nature, 293:265-270 (1981). |
Colbere-Garapin et al., "A New Dominant Hybrid Selective Marker for Higher Eukaryotic Cells", J. Mol. Biol., 150-1-14 (1981). |
Colot et al. EMBO Journal 6(12): 3559-3564 (1987). * |
Comai et al, "An Altered aroA Gene Product Confers Resistance to the Herbicide Glyphosate", Science, vol. 221, pp. 370-371 (Jul. 22, 1983). |
Comai et al, "Expression in plants of a mutant aroA gene from Salmonella typhimurium, confers tolerance to glyphosate", Nature, vol. 317, pp. 741-744 (Oct. 24, 1985). |
Comai et al, Chemical Abstracts, 99:182, abstract 100292j (Sep. 26, 1983). |
Condit et al, Abstract: Multiple Viral Specific Transcripts from the Genome of Cauliflower Mosaic Virus, Miami Winter Symposium, Jan. 17-21, 1983. |
Davey et al., "Transformation in plants: potential and reality", (Abstract of Conference paper from University of Nottingham) (1982). |
De Greve et al, Nature vol. 300 pp. 752-754 Dec. 1982. * |
DeGreve et al., "Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene", J. Mol. Appl. Genet., 1(6):499-511 (1982). |
DeGreve et al., "Regeneration of normal and fertile plants that express octopine synthase, from tobacco crown galls after deletion of tumour-controlling functions", Nature, 300:752-754 (1982). |
Depicker et al, "Nopaline Synthase: Transcript Mapping and DNA Sequence", Journal of Molecular and Applied Genetics 1:561-573 (1982). |
Depicker et al, Journal of Molecular and Applied Genetics vol. 1 pp. 561-573 Dec. 1, 1982. * |
Depicker et al, Journal of Molecular and Applied Genetics vol. 1 pp. 561-573. * |
Depicker et al. Journal of Molecular and Applied Genetics 1(6): 561-573 (1982). * |
Depicker et al., "Plant Cell Transformation by Agrobacterium Plasmids", Proceedings of a symposium held Aug. 15-19, 1982 at the University of California, Davis published in Genetic Engineering of Plants: An Agricultural Perspective, Basic Life Sciences (Kosuge et al., eds. 1983), pp. 143-176 (1982). |
Derwent Abstract of Patent Publication WP8301176-K16 (1983). * |
Dewent Abstract of Patent Publication EP-27662-D19 (1981). * |
Dewent Abstract of Patent Publication EP-67553-K01 (1983). * |
Dix et al., "A Cell Line of Nicotiana sylvestris with Resistance to Kanamycin and Streptomycin", Molec. Gen. Genet., 157:285-290 (1977). |
Dudits et al., "Backfusion with Somatic Protoplasts as a Method in Genetic Manipulation of Plants", Acta boil. Acad. Sci. hung., 32(3-4):215-218 (1981). |
Dudits et al., "Plant Regeneration from Intergeneric Cell Hybrids", Plant Science Letters, 15:101-112 (1979). |
Dunsmuir et al. Journal of Molecular and Applied Genetics 2(3): 285-300 (1983). * |
Ellis et al. EMBO Journal 6(1): 11-16 (1987). * |
F. White et al, "Tumor induction of Agrobacterium rhizogenes involves the transfer of plasmid DNA to the plant genome," Proc. Natl. Acad. Sci. USA, 79:3193-3197 (1982). |
Facciotti et al. Bio/Technology 3: 241-246 (1985). * |
First Response of Monsanto Company to Opponents Notices of Opposition in the Opposition Proceedings of corresponding European Patent Application No. 84900782.8-2105/0131623, Nov. 1992. |
Fischer et al., "Structure and flanking regions of soybean seed protein genes", Cell, 29:651-660 (1982). |
Fitzgerald et al Cell vol. 24 pp. 251-260 Apr. 1981. * |
Fitzgerald et al, "The Sequence of 5″-AAUAAA-3″ Forms Part of the Recognition Site for Polyadenylation of Late SV40 mRNAs", Cell 24:251-260 (Apr. 1981). |
Flavell et al., "Selectable marker genes: safe for plants?", Bio/Technology, 10:141-144 (1992). |
Fraley et al, Proceedings of the National Academy of Science of the USA, 80(15): 4803-4807 (Aug. 1983). |
Fraley et al., "Use of a Chimeric Gene to Confer Antibiotic Resistance to Plant Cells", Proceedings of the Miami Winter Symposium, Jan. 1983, published in Advances in Gene Technology: Molecular Genetics of Plants and Animals (Ahmad et al., eds.), pp. 211-221 (1983). |
Franck et al. Cell 21: 285-294 (Aug. 1980). * |
Franck et al., "Nucleotide sequence of cauliflower mosaic virus DNA", Cell, 21:285-294 (1980). |
Gardner et al. Nucleic Acids Research 9(12): 2871-2888 (1981). * |
Gardner et al., "Plant Viral Vectors: CaMV As an Experimental Tool", Proceedings of a symposium held Aug. 15-19, 1982 at the University of California, Davis, published in Genetic Engineering of Plants: an Agricultural Perspective, Basic Life Sciences (Kosuge et al., eds. 1983), pp. 121-142 (1982). |
Gardner et al., "The complete nucleotide sequence of an infectious clone of cauliflower mosaic virus by M13mp7 shotgun sequencing", Nucleic Acids Res., 9:2871-2888 (1981). |
Garfinkel et al., "Genetic Analysis of Crown Gall: Fine Structure Map of the T-DNA by Site-Directed Mutagenesis", Cell, 27:143-153 (1981). |
Gelvin, S. Plant Molecular Biology 8: 355-359 (1987). * |
Geraghty et al., "The primary structure of a plant storage protein: zein", Nucl. Acids Res., 9(19):5163-5174 (1981). |
Geraglty et al, Nucleic Acids Res. vol. 9 pp. 5163-5174 1981. * |
Gerlach et al., "cDNA cloning and induction of the alcohol dehydrogenase gene (Adh1) of maize", PNAS USA, 79:2981-2985 (1982). |
Gleba et al., "Arabidobrassica": A Novel Plant Obtained by Protoplast Fusion, Planta, 149:112-117 (1980). |
Gnonenborn et al, "Propagation of foreign DNA in plants using cauliflower mosaic virus as vector", Nature 294:773-776 (1981). |
Goldsbrough et al. Mol. Gen. Genet. 202: 374-381 (1986). * |
Goodman et al, "Gene Transfer in Crop Improvement", Science 236:48-54 (1987). |
Gordon et al., "Current Developments in the Transformation of Plants", Proceedings of the Miami Winter Symposium, Jan. 1983, published in Advances in Gene Technology: Molecular Genetics of Plants and Animals (Ahmad et al., eds.) pp. 37-46 (1983). |
Guilley et al, "Transcription of Cauliflower Mosaic Virus DNA: Detection of Promoter Sequences, and Characterization of Transcripts," Cell 23:763-773 (1982). |
Guilley et al. Cell 30: 763-773 (Oct. 1982). * |
Hall et al., "The Phaseolin Family of Seed Protein Genes: Sequences and Properties", published in Advances in Gene Technology: Molecular Genetics of Plants and Animals, (Ahmad et al., eds.), pp. 349-367 (1983). |
Heidicker, G. Nucleic Acids Research 11(14): 4891-4906 (1983). * |
Herman et al. Molecular and Cellular Biology 6(12): 4486-4492 (Dec. 1986). * |
Hernalsteens, "The Agrobacterium tumefaciens Ti plasmid as a host vector system for introducing foreign DNA in plant cells", Nature, 287:654-656 (1980). |
Herrera-Estrella et al, "Expression of chimaeric genes transferred into plant cells using a Ti-plasmid-derived vector", Chemical Abstracts, vol. 99, 17349n (1983). |
Herrera-Estrella et al. Nature 303: 209-213 (May 1983). * |
Herrera-Estrella et al., "Chimeric genes as dominant selectable markers in plant cells", EMBO J., 2:987-995 (1983). |
Herrera-Estrella et al., "Expression of chimaeric genes transferred into plant cells using a Ti-plasmid-derived vector", Nature, 303:209-213 (1983). |
Hoffmann et al., "Arabidobrassica": Chromosomal recombination and morphogenesis in asymmetric intergeneric hybrid cells, Planta, 153:586-593 (1981). |
Hohn et al., "Cauliflower Mosaic Virus on Its Way to Becoming a Useful Plant Vector", in Current Topics in Microbiology and Immunology, 96:193-236 (1982) (Henle et al., eds.). |
Holländer et al, "The Site of the Inhibition of the Shikimate Pathway by Glyphosate" Plant Physiol., 66, 823-829 (1980). |
Holster et al., "The Use of Selectable Markers for the Isolation of Plant-DNA/T-DNA Junction Fragments in a Cosmid Vector", Molec. Gen. Genet., 185:283-289 (1982). |
Hoorykoss et al, Genetic Engineering Principle and Methods vol. 1, Plenum Press N.Y. pp. 151-179 (1979). * |
Howell et al., "Cloned Cauliflower Mosaic Virus DNA Infects Turnips (Brassica rapa)", Science, 208:1265-1267 (1980). |
Howell, et al, "Rescue of in vitro generated mutants of cloned cauliflower mosaic virus genome in infected plants", Nature 293:483-486 (1981). |
Hu et al. EMBO Journal 1(11): 1337-1342 (1982). * |
Hu et al., "Primary structure of a genomic zein sequence of maize", EMBO J., 1:1337-1342 (1982). |
Hyldig-Nielsen et al., "The primary structures of two leghemoglobin genes from soybean", Nucleic Acids Res., 10(2):689-701 (1982). |
J. L. Fox, "Plant molecular biology beginning to flourish", Chemical and Engineering News, pp. 33-44 (Jun. 22, 1981). |
J. Leemans et al, "Ti Plasmids and Directed Genetic Engineering" in Molecular Biology of Plant Tumors, G. Kahl and J. Schell (eds.) pp. 537-545 (Academic Press NY 1982). |
J. Schell et al, Abstract from "Advances in Gene Technology: Molecular Genetics of Plants and Animals" distributed to attendees at the fifteenth Miami Winter Symposium, Jan. 17-21, 1983. (Oral presentation corresponding to patent made on Jan. 18, 1983). |
J.D. Kemp et al, Chemical Abstracts, 101:176, abstract 18452n (Jul. 3, 1984). |
Jimenez et al., "Expression of a transposable antibiotic resistance element in Saccharomyces", Nature, 287:869-871 (1980). |
Jones et al. EMBO Journal 4(10): 2411-2418 (1985). * |
Kaulen et al. EMBO Journal 5(1): 1-8 (1986). * |
Keith et al. EMBO Journal 5(10): 2419-2425 (1986). * |
Kemp et al., "Agrobacterium-Mediated Transfer of Foreign Genes into Plants", in Genetic Engineering: Application to Agriculture (Owens, ed.), pp. 215-228 (1983). |
Kim et al., "A 20 nucleotide upstream element is essential for the nopaline synthase (nos) promoter activity", Plant Molecular Biology, 24:105-117 (1994). |
Kleinhofs and Behki, "Prospects for Plant Genome Modification by Non-Conventional Methods", Ann. Rev. Genet. 11: 79-101 (1977). |
Komarnitsky et al., "Fraction I Protein Analysis of Parasexual Hybrid Plants Arabidopsis thaliana + Brassica campestris", Plant Cell Reports, 1:67-68 (1981). |
Koncz et al, "A simple method to transfer, integrate and study expression of foreign genes, such as chicken ovalbumin and α-actin in plant tumors", The EMBO Journal, vol. 3, No. 5:1029-1037 (1984). |
Kridl et al. Gene 28: 113-118 (1984). * |
Kridl et al., "Nucleotide sequence analysis of a zein genomic clone with a short open reading frame", Gene, 28:113-118 (1984). |
Lamppa et al. Nature 316: 750-752 (1985). * |
Larkin et al, International Congress of Plant Tissue and Cell Cultivation Abstract, Tokyo Jul. 11-16, 1982. |
Lebeurier et al., "Infectivities of native and cloned DNA of cauliflower mosaic virus", Gene, 12:139-146 (1980). |
Lee et al., "Control of tuber protein synthesis in potato", Plant Molecular Biology, Arco Solar-UCLA Symposium, Apr. 16-22, 1983, pp. 355-365. |
Leeman et al, Molecular Biology of Plant Tumors, Academy Press N. Y. pp. 537-545 (1982). * |
Leemans et al, J. Mol. Appl. Gen vol. 1 pp. 149-164 (1981). * |
Leemans et al., "Genetic identification of functions of TL-DNA transcripts in octopine crown galls", EMBO J., 1(1):147-152 (1982). |
Leemans et al., "Site-Specific Mutagenesis of Agrobacterium Ti Plasmids and Transfer of Genes to Plant Cells", J. Molec. Appl. Genet., 1(2):149-164 (1981). |
Leemans, "Ti to tomato, tomato to marker", Bio/Technology, 11:S22-S26 (1993). |
Leemans, Universite Libre de Bruxelles, Thesis, 1-25; 114-125 (1982). |
Lewin, B., ed. pp. 174-194 In: Genes, John Wiley and Sons: New York (1983). * |
Liu et al., "Agrobacterium Ti plasmid indoleacetic acid gene is required for crown gall oncogenesis", Proc. Natl. Acad. Sci. USA, 79:2812-2816 (1982). |
Llewellyn et al. pp. 593-607 In: Molecular Form and Function of the Plant Genome, van Vloten-Doting et al, eds., Plenum Publishing: New York (1985). * |
Lorz et al, "Transformation Studies Using Synthetic DNA Vectors Coding for Antibiotic Resistance," abstract of talk presented at the International Congress of Plant Tissue and Cell Culture, Jul. 11-16, 1982 in Tokyo. |
M. Van Montagu & J. Schell, "The Ti Plasmids of Agrobacterium", Current Topics in Microbiology and Immunology, 96:237-254 (1982). |
M.D. Chilton et al, "Tailoring the Agrobacterium Ti Plasmid as a Vector for Plan Genetic Engineering", Stadler Symposium (U. of Mo., Columbia) 13: 39-52 (1981). |
M.D. Chilton, "Agrobacterium Ti Plasmid as Tool for Genetic Engineering in Plants" p. 23, in Genetic Engineering of Osmoregulation, edited by D.W. Rains et al (Plenum Publ., NY 1980). |
Maliga et al., "Restoration of Morphogenic Potential in Nicotiana by Somatic Hybridisation", Molec. Gen. Genet., 157:291-296 (1977). |
Mar. 7, 1997 Decision of the Technical Board of Appeal 3.3.4. In the Opposition Appeal Proceedings T 0387/94-334 of corresponding European Patent Application No. 84900782.8-2106/0131623. |
Marks et al., "Molecular Structure and Expression of Maize Zein Genes", published in Advances in Gene Technology: Molecular Genetics of Plants and Animals, pp. 369-381 (1983). |
Marx, "Ti Plasmids as Gene Carriers: When suitably modified, tumor-inducing plasmids can transfer genes into plant cells from which normal plants may be regenerated", Science, 216:1305 (1982). |
Marx, J. Science 216: 1305 (Jun. 1982). * |
Matzke et al, J. Mol. Appl. Gen. vol. 1 pp. 39-49 (1981). * |
Matzke et al. EMBO Journal 3(7): 1525-1531 (1984). * |
Matzke et al., "Site-Specific Insertion of Genes into T-DNA of the Agrobacterium Tumor-Inducing Plasmid: An Approach to Genetic Engineering of Higher Plant Cells" J. Molec. Appl. Genet., 1:39-49 (1981). |
Matzke et al., "Transcription of a zein gene introduced into sunflower using a Ti plasmid vector", EMBO J., 3:1525-1531 (1984). |
McKnight et al., "Isolation of Mapping of Small Cauliflower Mosaic Virus DNA Fragments Active as Promoters in Escherichia coli", J. Virol., 37(2):673-682 (1981). |
Meagher et al., "A Model for a Molecular Cloning System in Higher Plants: Isolation of Plant Viral Promotors", NATO Advance Study Institute Series, 29:63-75 (1980). |
Meagher et al., "Plant actin is encoded by diverse multigene families", Advances in Gene Technology: Molecular Genetics of Plants and Animals, Miami Winter Symposia, 20:171-187. |
Melchers et al., "Somatic Hybrid Plants of Potato and Tomato Regenerated from Fused Protoplasts", Carlsberg Res. Commun., 43:203-218 (1978). |
Messing et al. pp. 211-227 In: Genetic Engineering of Plants, An Agricultural Perspective, Kosuge et al, eds., Plenum Press: New York (1983). * |
Messing et al., "Plant gene structure", Proceedings of Symposium held Aug. 15-19, 1982 at the University of California, Davis, published in Basic Life Sciences, 26:211-227 (1983). |
Messing et al., Proceedings of a symposium held Aug. 15-19, 1982 at the University of California, Davis published in Genetic Engineering of Plants: An Agricultural Perspective, Basic Life Sciences, 26:211-227 (Kosuge et al., eds. 1983) (1982). |
Messing, Joachim, Expert Opinion for presenting to the European Patent Office in the Opposition Appeal Proceedings T 0387/94-334 (corresponding European Patent Aplication) (1997). |
Mulligan et al., "Expression of a Bacterial Gene in Mammalian Cells", Science, 209:1422-1427. |
Mulligan et al., "Selection for animal cells that express that Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase", Proc. Natl. Acad. Sci. USA, 75(4):2072-2076 (1981). |
Mulligan et al., "Synthesis of rabbit beta-globin in cultured monkey kidney cells following infection with a SV40 beta-globin recombinant genome" Nature, 277:108-114 (1979). |
Mulligan et al., "Synthesis of rabbit β-globin in cultured monkey kidney cells following infection with a SV40 β-globin recombinant genome" Nature, 277:108-114 (1979). |
Murai et al. Science 222: 476-482 (1983). * |
Murai et al., "Phaseolin gene from bean is expressed after transfer to sunflower via tumor-inducing plasmid vectors", Science, 222:476-482 (1983). |
Nagy et al. EMBO Journal 4(12): 3063-3068 (1985). * |
Newell et al., "Successful Wide Hybridization Between the Soybean and a Wild Perennial Relative, G. tomentella Hayata", Crop Science, 22:1062-1065 (1982). |
Notice of Opposition of Agricultural Genetics Co. Ltd. in the Opposition Proceedings of corresponding European Patent Application No. 84900782.8-2105/0131623, (Jun. 1991). |
Notice of Opposition of BIOCEM in the Opposition Proceedings of corresponding European Patent Application No. 84900782.8-2105/0131623, (Jun. 1991). |
Notice of Opposition of ICI Seeds in the Opposition Proceedings of corresponding European Patent Application No. 84900782.8-2105/0131623, (Jun. 1991). |
Notice of Opposition of Max-Planck-Gesellschaft zur in the Opposition Proceedings of corresponding European Patent Application No. 84900782.8-2105/0131623. (Jun. 1991). |
Notice of Opposition of Mogen International N.V. in the Opposition Proceedings of corresponding European Patent Application No. 84900782.8-2105/0131623, (Jun. 1991). |
Notice of Opposition of Pioneer Hi-Bred International, Inc. in the Opposition Proceedings of corresponding European Patent Application No. 84900782.8-2105/0131623, (Jun. 1991). |
Notice of Opposition of Unilever PLC in the Opposition Proceedings of corresponding European Patent Application No. 84900782.8-2105/0131623, (Jun. 1991). |
Nov. 3, 1994 Interlocutory Decision of the Opposition Division in the Opposition Proceedings of corresponding European Patent Application No. 84900782.8-2105/0131623. |
O'Hare et al., "Transformation of mouse fibroblasts to methotrexate resistance by a recombinant plasmid expression a prokaryotic dihydrofolate reductase", PNAS, 78(3):1527-1531 (1981). |
Old et al., Principles of Gene Manipulation: An Introduction to Genetic Engineering, 1st ed.(Carr et al., eds.) University of California Press, vol. 2, pp. 9-23 (1980). |
Old et al., Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2nd ed.(Carr et al., eds.), University of California Press, vol. 2, pp. 121-210 (1981). |
Olszewski et al., "A Transcriptionally Active, Covalently Closed Minichromosome of Cauliflower Mosaic Virus DNA Isolated from Infected Turnip Leaves", Cell, 29:395-402 (1982). |
Ooms et al, "Octopine Ti-Plasmid Deletion Mutants of Agrobacterium tumefaciens with Emphasis on the Right Side of the T-Region", Plasmids, 7:15-29 (1982). |
Ooms et al, Plasmid 1, 15-29 1982. * |
Ooms et al., "Crown gall plant tumors of abnormal morphology, induced by Agrobacterium tumefaciens carrying mutated octopine Ti plasmids: analysis of T-DNA functions", Gene, 14:33-50 (1981). |
Otten et al, Mol. Gen. Genet. vol. 183 pp. 209-213 (1981). * |
Otten et al., "Mendelian Transmission of Genes Introduced into Plants by the Ti Plasmids of Agrobacterium tumefaciens", Molec. Gen. Genet., 183:209-213 (1981). |
P. J. J. Hooykaas et al, "Agrobacterium Tumor Inducing Plasmids: Potential Vectors for the Genetics Engineering of Plants" Genetic Enqnq Principles and Methods, 1:151 edited by J.K. Setlow and A. Hollaender (Plenum Press, NY, 1979). |
P.M. Gresshoff; "Growth Inhibition by Glyphosate and Reversal of its Action by Phenylalanine and Tyrosine", Aust. J. Plant Physiol., 6, 177-85 (1979). |
Peacock et al., "Gene transfer in maize: controlling elements and the alcohol . . . ", Advances in Gene Technology: Molecular Genetics of Plants and Animals, Miami Winter Symposia, vol. 20, pp. 311-325 (1983). |
Pedersen et al., "Cloning and sequence analysis reveal structural variation among related zein genes in maize", Cell, 29:1015-1026 (1982). |
Portetelle et al., Annales De Gembloux, 87e annee; 3e trimestre, No. 3:101-123 (1981). |
Poulsen et al., "Peptide Mapping of the Ribulose Bisphosphate Carboxylase Small Subunit From the Somatic Hybrid of Tomato and Potato", Carlsberg Res. Commun., 45:249-267 (1980). |
Ream et al, "Crown gall disease and prospects for genetic manipulation in plants", Science, 218:854-859 (Nov. 26, 1982). |
Rosahl et al. EMBO Journal 6(5): 1155-1159 (1987). * |
Schell et al, "The Use of Ti Plasmids as Gene Vectors for Plants", Genetic Engineering to Biotechnology—The Critical Transition, p. 41-53 (1982), Proceedings of a symposium in Rome, Italy Sep. 20-23, 1981. |
Schell et al, From Genetic Engineering to Biotechnology-The Critical Transistors, El. Whelon et al, Wiley & Sons Pub pp. 41-52 (May 21, 1982). * |
Schell et al, Genetic Engineering to Biotechnology-The Critical Transition, John Wiley & Sons Ltd. pp. 41-53, Proceedings of a Symposium in Rome Italy Sep. 20-23, 1981. * |
Schell et al., "The Ti Plasmids as Natural and as Practical Gene Vectors for Plants", Bio/Technology, Apr. 1983, pp. 175-180 (1983). |
Schell et al., "Ti Plasmids as Experimental Gene Vectors for Plants", Proceedings of the Miami Winter Symposium, Jan. 1983, published in Advances in Gene Technology: Molecular Genetics of Plants and Animals (Ahmad et al., eds.), pp. 191-209 (1983). |
Schell et al., Abstract of "Plant cell transformation and genetic engineering", published in Plant Improvement and Somatic Cell Genetics (Vasil et al., eds.), Academic Press, NY, pp. 255-276 (1982). |
Schell et al., Abstract of "Transfer of genes into plants via the Ti-plasmid of Agrobacterium tumifaciens", Proceedings of a conference held Jul. 3-7, 1978 in Wageningen, Netherlands, published in Broadening the Genetic Base of Crops (Harten et al., eds.), pp. 255-276 (1979). |
Schernthaner et al. EMBO Journal 7(5): 1249-1255 (1988). * |
Schiller et al., "Restriction Endonuclease Analysis of Plastid DNA from Tomato, Potato and Some of Their Somatic Hybrids", Mol. Gen. Genet., 186:453-459 (1982). |
Schoffl et al. EMBO Journal 4(5): 1119-1124 (1985). * |
Schoffl et al., "An analysis of mRNAs for a group of heat shock proteins of soybean using cloned cDNAs", J. Mol. Appl. Genet., 1:301-314 (1982). |
Schönbrunn et al, "Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail", PNAS, vol. 98, No. 4, pp. 1376-1380 (Feb. 13, 2001). |
Schroeder et al., "Ti-Plasmids: Genetic Engineering of Plants", in Plant Cell Culture in Crop Improvement (Sen et al., eds.), Plenum Press, NY, pp. 287-297 (1983). |
Seeburg, P.H. et al, "Efficient Bacterial Expression of Bovine and Porcine Growth Hormones," DNA 2(1): 37 (1983). |
Sengupta-Gopalan et al. Proc. Natl. Acad. Sci. USA 82: 3320-3324 (1985). * |
Shah et al, "Genes Encoding Actin in Higher Plants: Intron Positions Are Highly Conversed But the Coding Sequences Are Not", J. Mol. Appl. Genet. 2:111-126 (1983). |
Shah et al., "Complete nucleotide sequence of a soybean actin gene", Proc. Natl. Acad. Sci. USA, 79:1022-1026 (1982). |
Shaw et al, "A general method for the transfer of cloned genes to plant cells", Gene 23:315-330 (1983). |
Shaw et al. Nucleic Acids Research 12(20): 7831-7846 (1984). * |
Simpson et al, "DNA from the A6S/2 Crown Gall Tumor Contains Scrambled Ti-Plasmid Sequences near It Junctions with Plant DNA", Cell, 29:1005-1014 (1982). |
Simpson et al, Cell vol. 29 pp. 1005-1014 Jul. 1982. * |
Slightom et al., "Complete nucleotide sequence of a French bean storage protein gene: Phaseolin", PNAS USA, 80:1897-1901 (1983). |
Southern, P.J and P. Berg, "Transformation of Mammalian Cells in Antibiotic Resistance with Bacterial Gene Under Control of the SV40 Early Region Promoter," J. Mol. Appl. Gen. 1: 327-341 (1982). |
Stalker et al., "A Single Amino Acid Substitution in the Enzyme 5-Enolpyruvylshikimate-3-phosphate Synthase Confers Resistance to the Herbicide Glyphosate", The Journal of Biological Chemistry, vol. 260, No. 8, pp. 4724-4728 (Apr. 25, 1985). |
Stougaard et al. Proc. Natl. Acad. Sci. USA 84: 5754-5757 (1987). * |
Sun et al., "Intervening sequences in a plant gene-comparison of the partial sequence of cDNA and genomic DNA of French bean phaseolin", Nature, 289:37-41 (1981). |
Thomashow et al, "Integration and Organizaion of Ti Plasmid Sequence n Crown Gall Tumors" Cell, 19:729-739 (1980). |
Thomoshow et al, Cell vol. 19 pp. 729-739 Mar. 1980. * |
Timko et al., "Nuclear genes encoding the constituent polypeptides of the light-harvesting chlorophyll A/B-protein complex . . . ", Plant Molecular Biology, Arco Solar-UCLA Symposium, Apr. 16-22, 1983, pp. 403-412. |
Tuite et al, "Regulated high efficiency expression of human interferon-alpha in Saccharomyces cerevisiae," The EMBO Journal, 1:603-608 (1982). |
Ursic et al., "A New Antibiotic With Known Resistance Factors, G418, Inhibits Plant Cells", Biochem. Biophys. Res. Commun., 101(3):1031-1037 (1981). |
Van Montogu et al, Current Topics in Microbiology and Immunology, vol. 96, Springer Verlag pp. 238-254 (1982). * |
VanSlogteron et al, Chemical Abstracts, 97: 182, abstract 139517v (Oct. 25, 1982). |
Velten et al., "TR genes involved in agropine production", Molecular Genetics of the Bacteria-Plant Interaction, Verlag, Berlin, Puhler, A. (ed.), pp. 303-312 (1983). |
Watson, Molecular Biology of the Gene, 3rd ed., W.A. Benjamin, Inc., Menlo Park, California, pp. 379-495 (1977). |
White et al, PNAS USA vol. 79 pp. 3193-3197 May 1982. * |
Wiborg et al. Nucleic Acids Research 10(11): 3487-3494 (1982). * |
Wiborg et al., "The nucleotide sequences of two leghemoglobin genes from soybean", Nucleic Acids Res., 10:3487-3494 (1982). |
Willmitzer et al., "DNA from Ti plasmid present in nucleus and absent from plastids of crown gall plant cells", Nature, 287:359-361 (1980) (Nov. 1992). |
Willmitzer et al., "The TL-DNA in octopine crown-gall tumours codes for seven well-defined polyadenylated transcripts", EMBO J., 1(1):139-146 (1982). |
Wimpee et al., "Sequence heterogeneity in the RuBP carboxylase small subunit gene family of Lemna gibba", Plant Molecular Biology, Arco Solar-UCLA Symposium, Apr. 16-22, 1983, pp. 391-401. |
Yang et al., "Revertant seedlings from crown gall tumors retain a portion of the bacterial Ti plasmid DNA sequences", Proc. Natl. Acad. Sci. USA, 78(7):4151-4155 (1981). |
Zamboyski et al, J. Molecular and Applied Genetics vol. 1 pp. 361-370 (1982). * |
Zambrogski et al, Journal of Molecular and Applied Genetics vol. 1 pp. 361-370 Jun. 1, 1982. * |
Zambryski et al, "Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity", The EMBO Journal, 2(12):2143-2150, (1980). |
Zambryski et al, "Tumor Induction by Agrobacterium tumefacians: Analysis of Boundaries of T-DNA", J. Molecular and Applied Genetics 1:361-370 (1982). |
Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11236347B2 (en) | 2015-08-28 | 2022-02-01 | Pioneer Hi-Bred International, Inc. | Ochrobactrum-mediated transformation of plants |
US10894812B1 (en) | 2020-09-30 | 2021-01-19 | Alpine Roads, Inc. | Recombinant milk proteins |
US10947552B1 (en) | 2020-09-30 | 2021-03-16 | Alpine Roads, Inc. | Recombinant fusion proteins for producing milk proteins in plants |
US10988521B1 (en) | 2020-09-30 | 2021-04-27 | Alpine Roads, Inc. | Recombinant milk proteins |
US11034743B1 (en) | 2020-09-30 | 2021-06-15 | Alpine Roads, Inc. | Recombinant milk proteins |
US11072797B1 (en) | 2020-09-30 | 2021-07-27 | Alpine Roads, Inc. | Recombinant fusion proteins for producing milk proteins in plants |
US11142555B1 (en) | 2020-09-30 | 2021-10-12 | Nobell Foods, Inc. | Recombinant milk proteins |
US11401526B2 (en) | 2020-09-30 | 2022-08-02 | Nobell Foods, Inc. | Recombinant fusion proteins for producing milk proteins in plants |
US11685928B2 (en) | 2020-09-30 | 2023-06-27 | Nobell Foods, Inc. | Recombinant fusion proteins for producing milk proteins in plants |
US11840717B2 (en) | 2020-09-30 | 2023-12-12 | Nobell Foods, Inc. | Host cells comprising a recombinant casein protein and a recombinant kinase protein |
US11952606B2 (en) | 2020-09-30 | 2024-04-09 | Nobell Foods, Inc. | Food compositions comprising recombinant milk proteins |
US12077798B2 (en) | 2020-09-30 | 2024-09-03 | Nobell Foods, Inc. | Food compositions comprising recombinant milk proteins |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8273954B1 (en) | Genetically transformed plants | |
EP0131624B1 (en) | Plasmids for transforming plant cells | |
US5034322A (en) | Chimeric genes suitable for expression in plant cells | |
EP0131623B2 (en) | Chimeric genes suitable for expression in plant cells | |
US6174724B1 (en) | Chimeric genes suitable for expression in plant cells | |
CA1340736C (en) | Method for genetically modifying a plant cell for plant gene expression | |
US5102796A (en) | Plant structural gene expression | |
CA1313830C (en) | Glyphosate-resistant plants | |
JPH06315389A (en) | Plant transformation vector for promoting transc-ription in plant and bacteria | |
WO1987007299A1 (en) | Transformation and foreign gene expression in brassica species | |
EP0126546B2 (en) | Plant structural gene expression | |
JPH06339385A (en) | Method of promoting plant transcription using promoter of octopine t-dna | |
US8334139B1 (en) | Plasmids for transforming plant cells | |
JP2574130B2 (en) | Genetic modification of plant cells | |
CA1341254C (en) | Method for genetically modifying a plant cell for plant structural gene expression | |
DE3485921T2 (en) | PLASMIDES FOR TRANSFORMING PLANT CELLS. | |
CA1340713C (en) | Plant gene expression | |
AU733623B2 (en) | Method for transforming plants and vector therefor | |
JP2573797C (en) |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: MONSANTO TECHNOLOGY LLC, MISSOURI Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:PHARMACIA CORPORATION, FORMERLY KNOWN AS MONSATO COMPANY;REEL/FRAME:012350/0224 Effective date: 20010611 |
|
STCF | Information on status: patent grant |
Free format text: PATENTED CASE |
|
FPAY | Fee payment |
Year of fee payment: 4 |
|
MAFP | Maintenance fee payment |
Free format text: PAYMENT OF MAINTENANCE FEE, 8TH YEAR, LARGE ENTITY (ORIGINAL EVENT CODE: M1552); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY Year of fee payment: 8 |
|
MAFP | Maintenance fee payment |
Free format text: PAYMENT OF MAINTENANCE FEE, 12TH YEAR, LARGE ENTITY (ORIGINAL EVENT CODE: M1553); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY Year of fee payment: 12 |